Folate receptor targeted nanoparticle drug conjugates and uses thereof

ABSTRACT

The disclosure relates to nanoparticle drug conjugates (NDC) that comprise ultrasmall nanoparticles, folate receptor (FR) targeting ligands, and linker-drug conjugates, and methods of making and using them to treat cancer.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/105,995, filed on Oct. 27, 2020, U.S. Provisional Application No.63/116,393, filed on Nov. 20, 2020, U.S. Provisional Application No.63/117,110, filed on Nov. 23, 2020, U.S. Provisional Application No.63/155,043, filed on Mar. 1, 2021, U.S. Provisional Application No.63/222,181, filed on Jul. 15, 2021, U.S. Provisional Application No.63/242,201, filed on Sep. 9, 2021, and U.S. Provisional Application No.63/254,837, filed on Oct. 12, 2021, the contents of which are eachincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Targeted delivery of therapeutics (e.g., cytotoxic drugs) to cancercells is an emerging approach for cancer treatment. The toxicity of thedelivered therapeutics to healthy tissue or organs in a subject can begreatly reduced by the selective delivery of drugs to a targeted diseasearea, leading to improved therapeutic outcomes. Antibody drug conjugates(ADCs) are a popular platform for targeted drug delivery, whichtypically feature a highly toxic drug substance covalently attached to amonoclonal antibody that can target cancer, wherein the toxic drugsubstance is released upon targeting of the cancer. However, manychallenges remain with conventional targeted drug delivery platforms,such as ADCs, including difficulties in production, limitations in drugloading capacity, poor tumor penetration, and lack of ability toovercome tumor heterogeneity.

Cornell University and Memorial Sloan Kettering Cancer Center developedultrasmall sub-10 nm silica-organic hybrid nanoparticles, referred to asCornell prime dots (C'Dots), which have significant potential indiagnostics and therapeutic applications. For example, C'Dots can beconjugated with epidermal growth factor receptor inhibitors, e.g.,gefitinib, which is a cancer-targeted agent that inhibits cancer growth(WO 2015/183882 A1). However, the mechanism of action (MOA) of EGFRinhibitors requires active binding to the epidermal growth factorreceptor, so a continuous high concentration of the payload in thetargeted cancer cell is required to effectively inhibit cancer cellproliferation. This type of MOA is generally not compatible with thefast blood circulation half-life of C'Dots.

Folate receptor alpha (FRα), also known as FOLR1, has receivedsignificant attention from the scientific community as a potentialtarget for cancer therapy, and other isoforms of FR have also beenidentified as potential biological targets. See, e.g., Targeting FolateReceptor Alpha For Cancer Treatment, Cheung, A., et al. Oncotarget(2016) 7 (32):52553; Targeting the folate receptor: diagnostic andtherapeutic approaches to personalize cancer treatments, Ledermann, J.A. et al., Annals of Oncology (2015), 26:2034-2043; each of which areincorporated herein by reference in their entireties. Folate receptor(FR) is an ideal target for cancer therapy, as FR can be overexpressedin tumors, such as those of the ovary, endometrium, breast, colon, andlung, but its distribution in normal tissues is low and restricted.Emerging insights have suggested that FR may also exhibit cell-growthregulation and signaling functions, in addition to serving as a folatereceptor and transporter. These features together render FR anattractive therapeutic target.

Folic acid is transported into the cells by various mechanisms, and themost prevalent mechanism is mediation through folate receptors, of whichthere are four glycopeptide members (FR alpha [FOLR1], FR beta [FOLR2],FR gamma [FOLR3], and FR delta [FOLR4]). Among these four members, thealpha isoform (FR alpha or FRα) is a glycosylphosphatidylinositol(GPI)-anchored membrane protein with high affinity for binding andtransporting the active form of folate, 5-methyltetrahydrofolate (SMTF).The alpha isoform has been reported to be over-expressed in certainsolid tumors, for example, in ovarian cancer, fallopian tube cancer,primary peritoneum cancer, uterus cancer, kidney cancer, lung cancer,brain cancer, gastrointestinal cancer, and breast carcinomas. The alphaisoform is also over-expressed in certain hematological malignancies,which can be exploited for treatment of these malignancies, e.g., fortreatment of acute myeloid lymphoma (AML), including pediatric AML. Thislow and restricted distribution in normal tissues or cells, alongsideemerging insights into tumor-promoting functions and association ofexpression with patient prognosis, together render FRa an attractivetherapeutic target. Additionally, the beta isoform (FRβ) isoverexpressed in certain cancers, e.g., hematological malignancies suchas acute myeloid leukemia (AML) and chronic myelogenous leukemia (CML),providing the opportunity to develop targeted therapies for thesecancers.

Although many FR-targeted drug delivery platforms have been developedand tested for cancer treatment in the past, e.g., using both ADCs andsmall-molecule drug conjugates, none of them are successfully approvedfor clinical use due to their limited therapeutic outcome (EP 0624377A2, U.S. Pat. No. 9,192,682 B2, Leamon, et al., “Comparative preclinicalactivity of the folate-targeted Vinca alkaloid conjugates EC140 andEC145, Int. J. Cancer (2007) 121:1585-1592; Leamon et al., “Folate—VincaAlkaloid Conjugates for Cancer Therapy: A Structure-ActivityRelationships, Bioconjugate Chemistry (2014) 25:560-568; Scaranti, M.,et al. Exploiting the folate receptor a in oncology. Nat Rev Clin Oncol.(2020) 17: 349-359).

Therefore, successful development of a FR-targeted drug deliveryplatform remains highly desired.

SUMMARY OF THE INVENTION

The present disclosure provides a nanoparticle-drug conjugate (NDC)comprising: (a) a silica nanoparticle, and polyethylene glycol (PEG)covalently bonded to the surface of the nanoparticle; (b) a targetingligand comprising folic acid, or a derivative or salt thereof, whereinthe targeting ligand is attached to the nanoparticle directly orindirectly through a spacer group; and (c) a linker-payload conjugate,wherein: (i) the payload is exatecan; (ii) the linker-payload conjugateis attached to the nanoparticle directly or indirectly through a spacergroup; (iii) the linker is a protease-cleavable linker; and (iv) theexatecan is released upon cleavage of the linker.

This disclosure also relates to nanoparticle-drug-conjugates (NDCs)comprising: (a) a nanoparticle that comprises a silica-based core and asilica shell surrounding at least a portion of the core; polyethyleneglycol (PEG) covalently bonded to the surface of the nanoparticle, and afluorescent compound covalently encapsulated within the core of thenanoparticle; (b) a targeting ligand that binds to folate receptor (FR),wherein the targeting ligand comprises folic acid, or a folate receptorbinding derivative thereof, and wherein the targeting ligand is attachedto the nanoparticle directly or indirectly through a spacer group; (c) alinker-payload conjugate, wherein the payload is a cytotoxic agent;wherein the linker-payload conjugate is attached to the nanoparticledirectly or indirectly thorough a spacer group; wherein the cytotoxicagent is released upon cleavage of the linker; wherein the linker in thelinker-payload conjugate is a protease-cleavable linker; and wherein theNDC has an average diameter between about 1 nm and about 10 nm, e.g.,between about 3 nm and about 8 nm, or between about 3 nm and about 6 nm.The cytotoxic agent may be exatecan.

In the NDCs of the present disclosure, the average nanoparticle topayload ratio may range from 1 to 80, such as from 1 to 21 (e.g., 1 to13, or 1 to 12) and the average nanoparticle to targeting ligand ratiomay range from 1 to 50, such as from 1 to 25 (e.g., 1 to 11).

The NDCs of the present disclosure may have an average diameter ofbetween about 1 nm and about 10 nm, e.g., between about 5 nm and about 8nm, between about 3 nm and about 8 nm, or between about 3 nm and about 6nm.

The NDCs of the present disclosure may comprise any suitable dye ordetectable compound, such as a fluorescent compound. For example, in anNDC of the present disclosure, the fluorescent compound may be Cy5. Thefluorescent compound may be encapsulated within the nanoparticle (e.g.,covalently linked to the silica core). The NDCs of the presentdisclosure can comprise a targeting ligand that binds to a folatereceptor (FR). The targeting ligand may comprise folic acid or aderivative thereof. It should be understood that “folic acid” mayencompass an amide or an ester of folic acid, e.g., folic acid may beconjugated to the nanoparticle (or spacer group) at its carboxylterminus via an amide or ester bond. For example, “folic acid” may referto the folic acid amide present in the exemplary NDC illustrated in FIG.1 .

The NDCs of the present disclosure may comprise structure (S-1):

wherein Payload comprises exatecan; Linker comprises aprotease-cleavable linker; and the silicon atom is a part of thenanoparticle.

For example, the NDCs of the present disclosure may comprise structure(S-1a):

wherein the silicon atom is a part of the nanoparticle.

The NDCs of the present disclosure may comprise Structure (S-2):

wherein Targeting Ligand is folic acid, or a folate receptor bindingderivative thereof, and the silicon atom is a part of the nanoparticle.

For example, the NDCs of the present disclosure may comprise structure(S-2a):

wherein the silicon atom is a part of the nanoparticle.

The NDCs of the present disclosure may comprise a combination ofStructures (S-1) and (S-2). For example, the NDCs may comprise bothStructure (S-1a) and Structure (S-2a), e.g., as depicted in FIG. 1 .Structures S-1, S-1a, S-2, or S-2a may be present in the NDC at anydesired ratio, e.g., at a ratio disclosed herein.

The disclosure also relates to NDCs comprising a nanoparticle thatcomprises a silica-based core and a silica shell surrounding at least aportion of the core; polyethylene glycol (PEG) covalently bonded to thesurface of the nanoparticle; a fluorescent compound covalentlyencapsulated within the core of the nanoparticle; a targeting ligand,wherein the targeting ligand is folic acid; a linker-payload conjugate,wherein the linker-payload conjugate is a protease cleavable linker thatis capable of undergoing hydrolysis at a C-terminal end upon proteasebinding thereby releasing the payload from the nanoparticle, wherein theprotease comprises a serine protease or a cysteine protease, wherein thepayload in the linker-payload conjugate is exatecan, or an analog ofexatecan; and wherein the fluorescent compound is Cy5.

The disclosure also relates to NDCs comprising a nanoparticle thatcomprises a silica-based core and a silica shell surrounding at least aportion of the core; polyethylene glycol (PEG) covalently bonded to thesurface of the nanoparticle; a Cy5 dye covalently encapsulated withinthe core of the nanoparticle; a targeting ligand that binds to folatereceptor, wherein the targeting ligand is folic acid, and wherein thetargeting ligand is attached to the nanoparticle indirectly through aspacer group; a linker-payload conjugate, wherein the linker-payloadconjugate is attached to the nanoparticle indirectly through a spacergroup, wherein the linker-payload conjugate comprises a compoundcomprising the structure

and wherein the NDC has an average diameter between about 1 nm and about10 nm (e.g., between about 1 and about 6 nm).

This disclosure also relates to nanoparticle drug conjugates (NDC)comprising: (a) a silica nanoparticle that comprises a silica-based coreand a silica shell surrounding at least a portion of the core; andpolyethylene glycol (PEG) covalently bonded to the surface of thenanoparticle; (b) an exatecan-linker moiety comprising the structure ofFormula (NP-3):

wherein x is 4 and y is 9; and (c) a targeting ligand moiety comprisingthe structure of Formula (NP-2)

wherein x is 4 and y is 3, and wherein the exatecan-linker moiety andthe targeting ligand moiety are each conjugated to a surface of thenanoparticle. The NDC may comprise a fluorescent dye (e.g., Cy5)covalently encapsulated within the core of the nanoparticle.

This disclosure also provides a method of treating a folate receptor(FR)-expressing cancer (e.g., a folate receptor (FR)-expressing tumor),comprising administering to a subject in need thereof an effectiveamount of an NDC described herein. The method may include administrationof NDCs to the subject in need thereof intravenously. In the methods ofthe present disclosure, the subject may have a cancer selected from thegroup consisting of ovarian cancer, endometrial cancer, fallopian tubecancer, cervical cancer, breast cancer (including, e.g., HER2+ breastcancer, HR+ breast cancer, HR- breast cancer, and triple-negative breastcancer), lung cancer (e.g., non-small cell lung cancer (NSCLC),mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophagealcancer, colon cancer, rectal cancer, and stomach cancer), pancreaticcancer, bladder cancer, kidney cancer, liver cancer, head and neckcancer, brain cancer, thyroid cancer, skin cancer, prostate cancer,testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML),and chronic myelogenous leukemia (CML). The NDCs of the presentdisclosure may also be used for targeting tumor associated macrophages,which may be used as a means to modify the immune status of a tumor in asubject. The NDCs of the present disclosure may be used in a method oftreating an advanced, recurrent, or refractory solid tumor.

This disclosure provides use of an NDC for treating a folate receptor(FR)-expressing cancer (e.g., a folate receptor (FR)-expressing tumor).The use may include administration of NDCs intravenously to the subjectin need thereof. In the use of the NDC, the subject may have a cancerselected from the group consisting of ovarian cancer, endometrialcancer, fallopian tube cancer, cervical cancer, breast cancer(including, e.g., HER2+ breast cancer, HR+ breast cancer, HR- breastcancer, and triple-negative breast cancer), lung cancer (e.g., non-smallcell lung cancer (NSCLC), mesothelioma, uterine cancer, gastrointestinalcancer (e.g., esophageal cancer, colon cancer, rectal cancer, andstomach cancer), pancreatic cancer, bladder cancer, kidney cancer, livercancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer,prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g.,pediatric AML), and chronic myelogenous leukemia (CML). In the use ofthe NDC, the cancer may be an advanced, recurrent, or refractory solidtumor.

This disclosure provides NDCs for use in the manufacture of a medicamentfor treating a folate receptor (FR)-expressing cancer (e.g., a folatereceptor (FR)-expressing tumor). The use in the manufacture of amedicament may include administration of NDCs intravenously to thesubject in need thereof. The use in the manufacture of a medicament mayinclude administration of NDCs to a subject, wherein the subject has acancer selected from the group consisting of ovarian cancer, endometrialcancer, fallopian tube cancer, cervical cancer, breast cancer(including, e.g., HER2+ breast cancer, HR+ breast cancer, HR-breastcancer, and triple-negative breast cancer), lung cancer (e.g., non-smallcell lung cancer (NSCLC), mesothelioma, uterine cancer, gastrointestinalcancer (e.g., esophageal cancer, colon cancer, rectal cancer, andstomach cancer), pancreatic cancer, bladder cancer, kidney cancer, livercancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer,prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g.,pediatric AML), and chronic myelogenous leukemia (CML). The NDCs of thepresent disclosure may be used in the manufacture of a medicament fortreating an advanced, recurrent, or refractory solid tumors.

This disclosure also relates to a pharmaceutical composition comprisingan NDC and a pharmaceutically acceptable excipient. The pharmaceuticalcompositions disclosed herein may be used for treating a folate receptor(FR)-expressing cancer (e.g., a folate receptor (FR)-expressing tumor).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a representative chemical structure ofnanoparticle-drug conjugate (NDC).

FIG. 2 depicts a flow chart for the synthesis of an exemplaryfunctionalized nanoparticle (dibenzocyclooctyne (DBCO)-functionalizedC'Dot).

FIG. 3 depicts a flow chart for the synthesis of an exemplary NDC(FA-CDC) comprising a C'Dot functionalized with folic acid (FA) andexatecan.

FIG. 4 illustrates a representative UV-Vis absorbance spectrum of anexemplary functionalized nanoparticle (DBCO-functionalized C'Dot). Theabsorption peak at 648 nm correspond to the Cy5 dye that is covalentlyencapsulated within the core of the C'Dot. The absorption peaks around270 to 320 nm correspond to DBCO groups.

FIG. 5 illustrates a representative UV-Vis absorbance spectrum of anexemplary NDC (folic acid (FA)-functionalized C'Dot comprising exatecan(FA-CDC)). The absorption peak at 648 nm correspond to the Cy5 dye thatis covalently encapsulated within the core of C'Dot. The absorptionpeaks around 330 to 400 nm correspond to exatecan.

FIG. 6 depicts a fluorescence correlation spectroscopy (FCS) correlationcurve of an exemplary NDC (folic acid (FA)-functionalizedexatecan-linker conjugated C'Dot (FA-CDC)) that is fitted by asingle-modal FCS correlation function. Average hydrodynamic diameter wasobtained via fitting the FCS correlation curve.

FIG. 7 depicts a chromatogram showing the elution of an exemplary NDC(folic acid (FA)-functionalized exatecan-linker conjugated C'Dot(FA-CDC)) by a gel permeation chromatography (GPC). The elution ofFA-CDC (striped line under the curve) is compared to the elution time ofprotein standards with varying molecular weight (dashed line).

FIG. 8 depicts a reversed phase HPLC chromatogram of a purifiedexemplary NDC (folic acid (FA)-functionalized exatecan-linker conjugatedC'Dot (FA-CDC)) at 330 nm. This wavelength can be used to monitor boththe FA-CDC and impurities that may be present after the synthesis or dueto any degradation of the NDC.

FIG. 9 illustrates the UV-Vis absorbance spectra of an exemplaryexatecan-payload conjugate. Exatecan has an absorption maximum around360 nm.

FIGS. 10A-10B illustrate a representative HPLC chromatographs providinganalysis of an exemplary NDC prepared according to Example 3, that isconjugated with folic acid as targeting ligand and with exatecan as apayload (NDC prepared using the exatecan-linker conjugate precursor 202,from Example 1). FIG. 10A depicts a representative HPLC chromatograph at360 nm of the non-cleaved NDC showing a single peak at elution timearound 6.3 min which corresponds to the non-released payload retained onthe NDC. FIG. 10B depicts a representative HPLC chromatograph at 360 nmof a cleaved NDC, showing an additional peak at elution time around 3 to4 min which corresponds to the released exatecan payload. The area undercurve (AUC) of the released payload and the retained payload were usedto calculate the percentage of released payload.

FIGS. 11A-11C are plots illustrating a drug releasing analysis ofexemplary NDCs loaded with folic acid as targeting ligand and protease(cathepsin-B) cleavable exatecan-linker conjugates, at different timepoints after incubation with cathepsin-B. FIG. 11A depicts thereverse-phase HPLC chromatograph of NDC B at different time points afterincubation with Cathepsin-B. FIG. 11B depicts the reverse-phase HPLCchromatograph of NDC C at different time points after incubation withcathepsin-B. FIG. 11C depicts the reverse-phase HPLC chromatograph ofNDC D (prepared using the exatecan-linker conjugate precursor 202, fromExample 1) at different time points after incubation with cathepsin-B.

FIGS. 12A-12C are plots illustrating a drug releasing kinetics ofexemplary NDCs loaded with protease (cathepsin-B) cleavableexatecan-linker conjugates, at different time points after incubationwith cathepsin-B enzyme, and depicts the time for half of the payloadsto be released, i.e., T_(1/2). FIG. 12A depicts the T_(1/2) as 2.9 hoursfor NDC B. FIG. 12B depicts the T_(1/2) as 2.6 hours for NDC C. FIG. 12Cdepicts the T_(1/2) as 1.4 hours for NDC D.

FIG. 13 depicts the competitive binding of an exemplary NDC (folic acid(FA)-functionalized drug-linker conjugated C'Dot (FA-CDC)) in a FR alphapositive (KB) cell line, when compared with free folic acid.

FIG. 14 depicts the flow cytometry of representative NDCs (two folicacid (FA)-functionalized drug-linker conjugated C'Dot (FA-CDCs)) in KBcell line with varied folic acid ligand density (either an average of 0,12, or 25 folic acid molecules per nanoparticle). The exatecan-linkerconjugate precursor used to prepare each NDC used in the study isdescribed in Example 1 (Compound 202). Blocking in the blocking groupwas achieved using 1 mM of free folic acid. A CDC with no folic acid,but same amount of exatecan-linker conjugate, was used as the negativecontrol group.

FIG. 15 depicts the flow cytometry of representative NDCs (three folicacid (FA)-functionalized drug-linker conjugated C'Dots (FA-CDCs) in KBcell line with varied drug per particle ratio (DPR). The exatecan-linkerconjugate precursor used to prepare the NDCs used in the study isdescribed in Example 1 (Compound 202). Blocking in the blocking groupwas achieved using 1 mM of free folic acid. All FA-CDCs comprise between12 and 22 folic acid moieties. FA-CDCs with high drug-particle ratio(DPR) comprise between 35 and 50 exatecan-linker conjugate groups.FA-CDCs with medium DPR comprise between 17 and 25 exatecan-linkerconjugate groups. FA-CDCs with low DPR have between 5 and 10exatecan-linker conjugate groups. CDCs with no folic acid, and 17 to 25drug linkers, was used as the negative control group.

FIG. 16 depicts the flow cytometry of a representative NDC (folic acid(FA)-functionalized drug-linker conjugated C'Dot (FA-CDC)) at 1 nM thatwas pre-incubated with varied amounts of human plasma for 24 hours.Blocking in the blocking group was achieved with 1 mM of free folicacid. The exatecan-linker conjugate precursor used to prepare the NDCused in the study is described in Example 1 (Compound 202), to providean average of 25 exatecan molecules per nanoparticle. The average numberof folic acid ligands per nanoparticle was 15. CDC with no folic acid,but same amount of drug linkers was used as the negative control group.

FIG. 17 shows the confocal microscopy images of an exemplary NDC (folicacid (FA)-functionalized drug-linker conjugated C'Dot (FA-CDC), shown inExamples as NDC B) in KB (++++) and TOV-112D (−) cell lines at 1 hourand 24 hours. Blocking in the blocking group was achieved using 0.1 mMof free folic acid. The average number of folic acid ligands on theFA-CDC (NDC B) is 12, and the number of exatecan-linker conjugates is40). The lysosome was stained by using LysoTracker® Green, which is agreen-fluorescent dye for labeling and tracking acidic organelles inlive cells. With color images (not shown), the CDC appears red, thelysosome appears green, and the nucleus appears blue, due tofluorescence.

FIG. 18 is an image comparing the Z-stack confocal microscopic imagingof KB tumor spheroids treated with an exemplary folate-receptor(FR)-targeting NDC (NDC D, prepared according to Example 3 using theexatecan-linker conjugate precursor 202), a payload-free FR-targetingnanoparticle (FA-C'Dot), a FR-targeting ADC, or the correspondingpayload-free FR-targeting antibody, at 37° C. for 4 hours, followed bywashing. Scale bar: 200 um.

FIG. 19A depicts a representative maximum intensity projection (MIP)PET/CT imaging of healthy nude mice injected with ⁸⁹Zr-DFO-FA-CDC at 1,24, 48 and 72 hours post-injection.

FIG. 19B illustrates the biodistribution pattern of ⁸⁹Zr-DFO-FA-CDC inhealthy nude mice at 2 and 24 hour post-injection (n=3). Theexatecan-linker conjugate precursor used to prepare the NDC used in thestudy is described in Example 1 (Compound 202); the average number offolic acid ligands on each NDC (FA-CDC) is 12; and the average number ofexatecan-linker conjugates on each NDC is 25.

FIGS. 20A-20F depicts the in vivo tumor growth inhibition studies of sixexemplary folate receptor targeting NDCs (NDCs A-F) in KB tumor-bearingmice (n=7). NDC-A comprises about 19 drug-linker conjugate groups andabout 18 folic acid ligands per nanoparticle. NDC B comprises about 25drug-linker groups and about 15 folic acid ligands per nanoparticle. NDCC comprises about 19 drug-linker conjugate groups and about 13 folicacid ligands per nanoparticle. NDC D comprises about 25 drug-linkerconjugate groups and about 12 folic acid ligands per nanoparticle. NDC Ecomprises about 17 drug-linker conjugate groups and about 17 folic acidligands per nanoparticle. NDC F comprises about 23 drug-linker conjugategroups and about 20 folic acid ligands per nanoparticle.

FIGS. 21A-21B depict the IC₅₀ curves of an exemplary NDC inirinotecan-resistant and naïve KB cells, compared to non-conjugatedirinotecan. FIG. 21A shows the IC₅₀ curves of irinotecan in regular KBcells (naive cells), and in KB cells treated four times with irinotecan(irinotecan-resistant cells). FIG. 21B shows the IC₅₀ curves of theexemplary NDC in the naïve cells, and in the irinotecan-resistant cells.The exatecan-linker conjugate precursor used to prepare the exemplaryNDC of this study is described in Example 1 (Compound 202).

FIGS. 22A-22B depict the IC₅₀ curves of an exemplary NDC inexatecan-resistant and naive KB cells, compared to non-conjugatedexatecan. FIG. 21A shows the IC₅₀ curves of exatecan in regular KB cells(naive cells), and in KB cells treated four times or seven times withexatecan (exatecan-resistant cells). FIG. 22A shows the IC₅₀ curves ofthe exemplary NDC in the naive cells and in the exatecan-resistant cells(4-cycle and 7-cycle pretreatment). The exatecan-linker conjugateprecursor used to prepare the exemplary NDC of this study is describedin Example 1 (Compound 202).

FIG. 23 provides a table demonstrating the cytotoxicity of exemplaryfolate receptor targeting NDCs (“FA-CDC”) with varying drug-to-particleratios (DPRs), in different FR-alpha overexpressing cancer cell lines,compared to non-conjugated exatecan. The exatecan-linker conjugateprecursor used to prepare the exemplary NDCs of this study is describedin Example 1 (Compound 202).

FIG. 24 provides a table showing the cytotoxicity of an exemplary NDC invarious 3D patient-derived platinum-resistant tumor spheroids. Theexatecan-linker conjugate precursor used to prepare the exemplary NDC ofthis study is described in Example 1 (Compound 202).

FIGS. 25A-25D provide flow cytometry histograms demonstrating thespecific folate receptor (FR) alpha targeting capability of an exemplaryFR-targeting NDC (prepared according to Example 3, using theexatecan-linker conjugate precursor 202 of Example 1) to both theIGROV-1 (FR alpha positive human ovarian cancer) and the engineered AMLMV4;11 cell line that overexpresses FR alpha. FIG. 25A is the flowcytometry histogram of the FR targeting NDC (10 nM) and non-targetingNDC (negative control; 10 nM) in IGROV-1 cell line. FIG. 25B is the flowcytometry histogram of anti-FR alpha antibody-PE and isotype antibody-PE(negative control) in IGROV-1 cell line. FIG. 25C is the flow cytometryhistogram of the FR targeting NDC (10 nM) and non-targeting NDC(negative control; 10 nM) in engineered AML MV4;11 cell line thatoverexpresses FR alpha. FIG. 25D is the flow cytometry histogram ofanti-FR alpha antibody-PE and isotype antibody-PE (negative control) inengineered AML MV4;11 cell line that overexpresses FR alpha.

FIGS. 26A-26B are graphs illustrating the in vitro cytotoxic activity ofan exemplary NDC (prepared according to Example 3 using theexatecan-linker conjugate precursor of Example 1, Compound 202) inIGROV-1 (FR alpha positive human ovarian cancer) cell line (FIG. 26A)and MV4; 11 engineered AML MV4;11 cell line that overexpresses FR alpha(FIG. 26B) using non-targeted NDC as negative control.

FIG. 27 is a graph providing the bodyweight change of FR alphaoverexpressing AML mice over time after treatment with normal saline oran exemplary NDC (prepared according to Example 3, using theexatecan-linker conjugate precursor of Example 1, Compound 202) at threedifferent dose regimens (0.33 mg/kg, Q3Dx6 (denoted with squares); 0.50mg/kg, Q3Dx3 (denoted with diamonds); or 0.65 mg/kg, Q3Dx3 (denoted withtriangles)).

FIG. 28 provides images from in vivo bioluminescence imaging (BLI) of FRalpha overexpressing AML mice treated with normal saline or an exemplaryNDC (prepared according to Example 3, using the exatecan-linkerconjugate precursor of Example 1, Compound 202) at three different doseregimens (0.33 mg/kg, Q3Dx6); 0.50 mg/kg, Q3Dx3; or 0.65 mg/kg, Q3Dx3).

FIG. 29 is a graph providing the quantitative in vivo bioluminescenceimaging analysis of FR alpha overexpressing AML mice treated with normalsaline or an exemplary NDC (prepared according to Example 1, using theexatecan-linker conjugate precursor of Example 1, Compound 202) at threedifferent dose regimens (0.33 mg/kg, Q3Dx6); 0.50 mg/kg, Q3Dx3; or 0.65mg/kg, Q3Dx3).

FIG. 30 is a graph indicating the leukemia detected in bone marrowaspiration at Day 42 post-leukemia cell injection, obtained from micetreated with normal saline or an exemplary NDC (prepared according toExample 3, using the exatecan-linker conjugate precursor of Example 1,Compound 202) at three different dose regimens (0.33 mg/kg, Q3Dx6); 0.50mg/kg, Q3Dx3; or 0.65 mg/kg, Q3Dx3).

FIG. 31 is an illustration of the timeline used for preparing FR alphaoverexpressing AML mice and dosing the mice with an exemplary NDC(prepared according to Example 3, using the exatecan-linker conjugateprecursor of Example 1, Compound 202) at three different dose regimens(0.33 mg/kg, Q3Dx6); 0.50 mg/kg, Q3Dx3; or 0.65 mg/kg, Q3Dx3), andimaging the mice with bioluminescent imaging (BLI). Each day of dosingis denoted by a triangle (i.e., on days 46, 49, and 52 for all dosegroups, and also on days 55, 58, and 62 for the 0.33 mg/kg Q3Dx6 dosegroup).

FIG. 32 shows the confocal microscopy images of an exemplary NDC (folicacid (FA)-functionalized drug-linker conjugated C'Dot (FA-CDC), shown inExamples as NDC D, prepared using exatecan-linker conjugate precursor202) in KB (++++) and TOV-112D (−) cell lines after 1 hour and 24 hours.Blocking in the blocking group was achieved using 0.1 mM of free folicacid. The lysosome was stained by using LysoTracker® Green, which is agreen-fluorescent dye for labeling and tracking acidic organelles inlive cells. With color images (not shown), the CDC appears red, thelysosome appears green, and the nucleus appears blue, due tofluorescence.

FIGS. 33A-33B are graphs demonstrating the stability of exemplary NDCsprepared using methods disclosed herein. FIG. 33A compares the stabilityof an NDC produced using a diene-based functionalized nanoparticle(i.e., based on the protocol outlined in Example 3), and a comparativeNDC produced using an amine-based functionalized nanoparticle, in humanserum at 37° C., over 7 days. FIG. 33B compares the stability of the NDCproduced using a diene-based bifunctional precursor, and the comparativeNDC produced using an amine-based bifunctional precursor, in mouse serumat 37° C., over 7 days.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are nanoparticle drug conjugates (NDC), which comprisea nanoparticle (e.g., a silica nanoparticle, such as a multi-modalsilica-based nanoparticle) that allows conjugation to targeting ligandsand to cytotoxic payloads, for detection, prevention, monitoring, and/ortreatment of a disease, such as cancer.

This disclosure provides compositions and methods of using ananoparticle drug conjugate (NDC) comprising: a nanoparticle; atargeting ligand that binds to a folate receptor (e.g., folic acid, or aderivative or salt thereof), and a linker-payload conjugate, that maycomprise exatecan and a protease-cleavable linker.

The conjugation of both targeting ligands and linker-drug conjugates tothe nanoparticle can be achieved via a highly efficient “clickchemistry” reaction, which is fast, simple to perform, versatile, andresults in high product yields. The payload may be a cytotoxic agentcomprising exatecan, or a salt or analog thereof, that is attached tothe nanoparticle via a cleavable linker group. The cleavable linkergroup can be cleaved when the NDC is internalized in a cancer cell(e.g., in a tumor cell), such as in the endosome or lysosomalcompartment of the cell, causing release of the active cytotoxic agentfrom the NDCs. The cleavage may be catalyzed by a protease (e.g.,cathepsin B).

The NDCs disclosed herein provide an optimal platform for drug delivery,due in part to their physical properties. For example, the NDCs comprisenanoparticles that are ultrasmall in diameter (e.g., with averagediameter between about 1 nm and about 10 nm, such as between about 5 nmand about 8 nm) and benefit from enhanced permeability and retention(EPR) effects in tumor microenvironments, while retaining desiredclearance and pharmacokinetic properties.

The NDCs described herein have certain advantages over other drugdelivery platforms (e.g., ADCs such as FR-targeted ADCs, and FR-targetedsmall molecule drugs (e.g., chemotherapeutics)). For example, a singleNDC of the present disclosure may include up to about 80 drug moleculeson each nanoparticle (e.g., 80 exatecan molecules). In contrast, inconventional ADCs, only about 4 to 8 therapeutic/drug molecules can beattached to the antibody, and conventional FR-targeted small moleculedrugs are limited to only a single therapeutic/drug molecule. Thus, theNDCs described herein can carry at least 10 times more drug moleculesNDC, relative to conventional drug delivery platforms, and deliver arelatively higher drug load to cells.

While conventional folate receptor (FR)-targeting drug-deliveryplatforms, such as ADCs and FR-targeted small molecularchemotherapeutics, usually exhibit high potency in cancer cells withhigh receptor expression level, their efficacy in cancer cells withmedium or low FR expression level is limited. In contrast, the NDCs ofthe present disclosure can effectively target cancer cells with bothhigh and low FR expression levels and provide potent therapy for cancersthat have low FR expression (see, e.g., FIG. 23 and associated assaydescribed in the Examples).

Without wishing to be bound by any particular theory of mechanism, it isbelieved that, because the NDCs disclosed herein can include multipleFR-targeting ligands on a single nanoparticle, there is a multivalent oravidity effect on binding to several FRs on the cell surface. Incontrast, a single ADC generally can only bind to up to two FRs on thecell surface, and a single FR-targeted chemotherapy drug can only bindto one FR on the cell surface. Thus, the multivalent effect of theFR-targeted NDCs of the present disclosure can significantly enhance thebinding of NDC to cells that express FR, leading to improved targetingefficiency and therapeutic outcomes. This multivalent effect can alsorender the NDCs of the present disclosure suitable for treating cancersthat have low FR-expression, that cannot be effectively treated usingconventional FR-targeted drug delivery platforms, such as ADCs orFR-targeted chemotherapy drugs.

The efficacy of ADCs in solid tumor treatment is usually greatly limitedby their poor tumor penetration. In contrast, the FR-targeted NDCsdisclosed herein exhibit highly effective tumor penetration, permittingthe delivery of therapeutics throughout a tumor followingadministration, which improves therapeutic outcomes in treating solidtumors, relative to the use of ADCs.

The NDCs of the present disclosure have a smaller size than conventionaldrug delivery platforms, such as ADCs. Notably, the NDCs of the presentdisclosure are smaller than the particle size cut off for renalclearance, permitting the NDC to be renally clearable. As a result, NDCsthat are administered to a subject but do not enter a cancer cell (i.e.,non-targeted NDCs) can be rapidly cleared from the body via renalelimination. This target-and/or-clear approach reduces the toxicity ofNDCs as compared to conventional drug delivery platforms, such as ADCs,and prevents undesirable accumulation of the NDCs (or their payloads) inhealthy tissues or organs. The NDCs of the present disclosure exhibitimproved biodistribution than conventional drug delivery platforms, suchas ADCs, resulting in reduced side effect and toxicity. Nanoparticles

This disclosure relates to NDCs comprising a nanoparticle, such as asilica nanoparticle. The nanoparticle may comprise a silica-based coreand a silica shell surrounding at least a portion of the core.Alternatively, the nanoparticle may have only the core and no shell. Thecore of the nanoparticle may contain the reaction product of a reactivefluorescent compound and a co-reactive organo-silane compound. Forexample, the core of the nanoparticle may contain the reaction productof a reactive fluorescent compound and a co-reactive organo-silanecompound, and silica. In preferred aspects of the present disclosure,the nanoparticle is a core-shell particle.

The diameter of the core may be from about 0.5 nm to about 100 nm, fromabout 0.1 nm to about 50 nm, from about 0.5 nm to about 25 nm, fromabout 0.8 nm to about 15 nm, or from about 1 nm to about 8 nm. Forexample, the diameter of the core may be from about 3 nm to about 8 nm,or 3 nm to about 6 nm, e.g., the diameter of the core may be from about3 nm to about 4 nm, about 4 nm to about 5 nm, about 5 nm to about 6 nm,about 6 nm to about 7 nm, or about 7 nm to about 8 nm.

The shell of the nanoparticle can be the reaction product of a silicaforming compound, such as a tetraalkyl orthosilicate, for exampletetraethyl orthosilicate (TEOS). The shell of the nanoparticle may havea range of layers. For example, the silica shell may be from about 1 toabout 20 layers, from about 1 to about 15 layers, from about 1 to about10 layers, or from about 1 to about 5 layers. For example, the silicashell may comprise from about 1 to about 3 layers. The thickness of theshell may range from about 0.5 nm to about 90 nm, from about 0.5 nm toabout 40 nm, from about 0.5 nm to about 20 nm, from about 0.5 nm toabout 10 nm, or from about 0.5 nm to about 5 nm, e.g., about 1 nm, about2 nm, about 3 nm, about 4 nm, or about 5 nm. For example, the thicknessof the silica shell may be from about 0.5 nm to about 2 nm. The silicashell of the nanoparticle may cover only a portion of nanoparticle orthe entire particle. For example, the silica shell may cover about 1 toabout 100 percent, from about 10 to about 80 percent, from about 20 toabout 60 percent, or from about 30 to about 50 percent of thenanoparticle. For example, the silica shell may cover about 50 to about100 percent. The silica shell can be either solid, i.e., substantiallynon-porous, meso-porous, semi-porous, or the silica shell may be porous.The silica nanoparticle can be either solid, i.e., substantiallynon-porous, meso-porous, semi-porous, or the silica nanoparticle may beporous. In some embodiments, the nanoparticle is a non-mesoporousnanoparticle, e.g., a non-mesoporous silica nanoparticle, such as anon-mesoporous silica core-shell nanoparticle.

The surface of the nanoparticle may be modified to incorporate at leastone functional group. An organic polymer may be attached to thenanoparticle and can be modified to incorporate at least one functionalgroup by any known techniques in the art. The functional groups caninclude, but are not limited to, dibenzocyclooctyne (DBCO), maleimide,N-hydroxysuccinimide (NHS) ester, a diene (e.g., cyclopentadiene), anamine, or a thiol. For example, a bifunctional group comprising a silaneat one terminus, and a DBCO, maleimide, NHS ester, diene (e.g.,cyclopentadiene), amine, or thiol at the other terminus, may becondensed onto the surface of the silica nanoparticle via the silanegroup. The incorporation of the functional group can also beaccomplished through known techniques in the art, such as using “clickchemistry,” amide coupling reactions, 1,2-additions such as a Michaeladdition, or Diels-Alder (2+4) cycloaddition reactions. Thisincorporation allows attachment of various targeting ligands, contrastagents and/or therapeutic agents to the nanoparticle.

The organic polymers that may be attached to the nanoparticle include,but are not limited to, poly(ethylene glycol) (PEG), polylactate,polylactic acids, sugars, lipids, polyglutamic acid (PGA), polyglycolicacid, poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), andcombinations thereof. In preferred aspects of the present disclosure,the organic polymer is poly(ethylene glycol) (PEG).

In preferred aspects of the present disclosure, the surface of thenanoparticle is functionalized. For example, the surface of thenanoparticle can have functional groups other than those resulting fromthe synthesis of the nanoparticles (e.g., -OH groups (resulting fromterminal Si-OH groups on a nanoparticle surface) and PEG groups(resulting from Si-PEG groups on the nanoparticle surface). Suchfunctionalization and functionalization methods are known in the art.

The nanoparticle may comprise a non-pore surface and a pore surface. Inan embodiment, at least a portion of the individual nanoparticlenon-pore surface and at least a portion of the individual nanoparticlepore surface are functionalized. In an embodiment, at least a portion ofthe nanoparticle non-pore surface and the at least a portion of the poresurface have different functionalization. The pore surface is alsoreferred to herein as the interior surface. The nanoparticles may alsohave a non-pore surface (or non-porous surface). The non-pore surface isalso referred to herein as the exterior nanoparticle surface.

The pore surface (e.g., at least a portion of the pore surface) and/orthe non-pore surface (e.g., at least a portion of the non-pore surface)of the nanoparticle can be functionalized. For example, thenanoparticles can be reacted with compounds such that a functional groupof the compound is presented on (e.g., covalently bonded to) the surfaceof the nanoparticle. The surface can be functionalized with hydrophilicgroups (e.g., polar groups such as ketone groups, carboxylic acid,carboxylate groups, and ester groups), which provide a surface havinghydrophilic character, or hydrophobic groups (e.g., nonpolar groups suchas alkyl, aryl, and alkylaryl groups), which provide a surface havinghydrophobic character. Such functionalization is known in the art. Forexample, diethoxydimethylsilane (DEDMS) can be condensed on at least aportion of the pore surface such that the pore surface has hydrophobiccharacter, allowing increased loading performance of a hydrophobiccytotoxic payload relative to nanoparticles that are not functionalizedso.

In preferred aspects of the present disclosure, the surface of thenanoparticle is at least partially functionalized with polyethyleneglycol (PEG) groups. The attachment of PEG to the nanoparticle may beaccomplished by a covalent bond or a non-covalent bond, such as by ionicbond, hydrogen bond, hydrophobic bond, coordination, adhesive, andphysical absorption.

In certain aspects, the PEG groups are attached (e.g., covalentlyattached) to the surface of the nanoparticle. In a core-shellnanoparticle, the PEG groups are covalently bonded to the silica at thesurface of the shell via a Si-O-C bond and or to the silica in the core.In a core nanoparticle, the PEG groups are covalently bonded to thesilica in the core.

In preferred aspects, the nanoparticle is a core-shell nanoparticle,wherein the PEG groups are covalently bonded to the silica at thesurface of the shell via a Si-O-C bond. The PEG groups on thenanoparticle surface can prevent adsorption of serum proteins to thenanoparticle in a physiological environment (e.g., in a subject), andmay facilitate efficient urinary excretion and decrease aggregation ofthe nanoparticle (see, e.g., Burns et al. “Fluorescent silicananoparticles with efficient urinary excretion for nanomedicine”, NanoLetters (2009) 9(1):442-448).

The PEG groups may be derived from PEG polymer having a molecular weight(Mw) of 400 g/mol to 2000 g/mol, including all integer g/mol values andranges therebetween. In an embodiment, the PEG groups are derived fromPEG polymer having a Mw of 460 g/mol to 590 g/mol, which contain 6 to 9ethylene glycol units. In various embodiments, the nanoparticles are atleast 50%, at least 75%, at least 90%, or at least 95% functionalizedwith PEG groups. In an embodiment, the nanoparticles are functionalizedwith PEG groups with the maximum number of PEG groups such that, thepores remain accessible (e.g., the pores can be functionalized). In anembodiment, the pore surface is a silica surface having terminal silanol(Si-OH) groups.

A polyethylene glycol unit disclosed herein may be functionalized with afunctional group, for example, a “click chemistry” group, such asdibenzocyclooctyne (DBCO) or azide, a diene (e.g., cyclopentadiene), amaleimide, an NHS ester, an amine, a thiol, or an activated acetylenemoiety such as

While DBCO can be used, the functional group may also be another alkyne,such as another strained alkyne (e.g., DIBO or a derivative thereof, ora derivative of DBCO). Also, the functional group may be a nitrone or anitrile oxide.

Alternatively, or in addition to the foregoing, a functional group canbe introduced to an NDC without necessarily requiring a PEG group. Forexample, an NDC may be functionalized with a functional group such as a“click chemistry” group, e.g., dibenzocyclooctyne (DBCO) or azide; adiene (e.g., cyclopentadiene); a maleimide; an NHS ester; an amine; athiol; or an activated acetylene moiety such as

that may comprise any suitable linker, or may have no linker. While DBCOcan be used to functionalize the nanoparticle, the functional group mayalso be another alkyne, such as another strained alkyne (e.g., DIBO or aderivative thereof, or a derivative of DBCO). Also, the functional groupmay be a nitrone or a nitrile oxide.

For example, a DBCO-functionalized linker may be introduced to ananoparticle (e.g., a PEGylated C'Dot) by reacting the silane group on aDBCO-linker-silane compound with a silanol group on the surface of thenanoparticle (e.g., under the PEG layer on the C'Dot surface).Similarly, a diene-functionalized precursor (e.g.,cyclopentadiene-functionalized precursor) may be introduced to ananoparticle (e.g., a PEGylated C'Dot) by reacting the silane group on adiene-linker-silane or diene-silane precursor compound with a silanolgroup on the surface of the nanoparticle (e.g., under the PEG layer onthe C'Dot surface), followed by functionalizing the diene on thenanoparticle with a second precursor that comprises a reactive group(e.g., DBCO) via a dienophile. The linker group in theDBCO-linker-silane or diene-linker-silane can comprise any structure (orsub-structure), including but not limited to PEG, a carbon chain (e.g.,alkylene), a heteroalkylene group, or the like. The diene-functionalizedlinker covalently attached to the nanoparticle may be further modified,e.g., by reaction with a DBCO-functionalized group. For example, thediene-functionalized linker covalently attached to the nanoparticle maybe contacted with a DBCO-linker-maleimide compound (or other suitableDBCO-linker-dienophile), to form a cycloadduct between the diene andmaleimide, resulting in an NDC comprising DBCO groups attached to itssurface, e.g., using cycloaddition chemistry, such as a Diels-Aldercycloaddition.

Functionalization (e.g., with one of the aforementioned functionalgroups, such as DBCO or cyclopentadiene) facilitates the conjugation ofsuitably functionalized FR-targeting ligands and/or functionalized drugpayloads (such as azide-functionalized FR-targeting ligands and/orazide-functionalized drug payloads) to the nanoparticle by a couplingreaction, e.g., via click chemistry, (3+2) cycloaddition reactions,amide coupling, or Diels-Alder reaction. This functionalization approachalso improves the versatility of the formulation chemistry and thestability of the FR-targeted NDC constructs.

An advantage of the NDCs disclosed herein is that they can be preparedusing relatively stable linker or spacer groups, or precursors thereof.The linker or spacer groups, or their precursors, can avoid premature orundesired cleavage, which can occur using other linkers or precursors.For example, certain methods of functionalizing nanoparticles employamine-silane precursors (to provide amine-functionalized nanoparticles)that are modified at the amine groups to conjugate other moieties to thenanoparticle. However, the amine-silane precursors can be unstable andcan self-condense during reaction, causing undesired aggregation. Theaggregates can be very difficult to separate from the functionalizednanoparticles. Additionally, the amine groups on the surface of thenanoparticle can promote undesired reactivity, that may lead topremature release of the payload, or undesired release of the targetingligand.

The NDCs disclosed herein can be produced using relatively stableprecursors, and the NDCs are stable and highly pure. For example, thenanoparticles of the present NDCs can be prepared with a silane-dieneprecursor (such as a silane-cyclopentadiene precursor), to afford ananoparticle functionalized with one or more diene groups. The dienegroups may then be reacted with a second precursor, such as adienophile-containing precursor (e.g., a PEG-maleimide derivative, e.g.,a DBCO-PEG-maleimide), causing a stable cycloadduct to form. Theresulting functionalized nanoparticle, comprising the cycloadduct, mayoptionally be reacted with one or more subsequent precursors (such astargeting ligand precursors and/or payload-linker conjugate precursorsdescribed herein), to further functionalize the nanoparticle. Thediene-silane precursors, and the cycloadducts that are produced, do notexhibit the undesired qualities of other functionalized nanoparticles,e.g., they have relatively high serum stability, can be produced in highyield and purity (e.g., free of aggregated precursor). See, e.g., FIGS.33A-33B. Additionally, as this nanoparticle functionalization approachis highly modular, any desired ratio of payload, targeting ligand, orotherwise, can be introduced to the nanoparticle. Examples of preparingnanoparticles using these methods, and their benefits, are provided inthe Examples.

The NDCs of the present disclosure may comprise a structure of Formula(NP):

wherein x is an integer of 0 to 20, e.g., 4; wherein the silicon atom isa part of the nanoparticle; and wherein the

adjacent to the triazole moiety denotes a point of attachment to atargeting ligand or payload-linker conjugate, either directly orindirectly, e.g., via a linker or spacer group, e.g., a PEG moiety. Forexample, the attachment may be to a linker or spacer group, e.g., thelinker of a linker-payload conjugate, or a linker or spacer group of afolate receptor targeting ligand, e.g., a PEG moiety. The NDCs of thepresent disclosure may be prepared from diene (e.g., cyclopentadiene)functionalized nanoparticles, e.g., by conjugating a linker moiety(e.g., a linker comprising a dienophile, such as maleimide) to the dienewith a cycloaddition reaction.

The silica shell surface of the nanoparticles can be modified by usingknown cross-linking agents to introduce surface functional groups.Crosslinking agents include, but are not limited to, divinyl benzene,ethylene glycol dimethacrylate, trimethylol propane trimethacrylate,N,N′-methylene-bis-acrylamide, alkyl ethers, sugars, peptides, DNAfragments, or other known functionally equivalent agents.

In order to permit the nanoparticle to be detectable by not only opticalimaging (such as fluorescence imaging), but also other imagingtechniques, such as positron emission tomography (PET), single photonemission computed tomography (SPECT), computerized tomography (CT), andmagnetic resonance imaging (MRI), the nanoparticle may also beconjugated to a contrast agent, such as a radionuclide.

The nanoparticles may incorporate any suitable fluorescent compound,such as a fluorescent organic compound, a dye, a pigment, or acombination thereof. Such fluorescent compounds can be incorporated intothe silica matrix of the core of the nanoparticle. A wide variety ofsuitable chemically reactive fluorescent dyes/fluorophores are known,see for example, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES ANDRESEARCH CHEMICALS, 6^(th) ed., R. P. Haugland, ed. (1996). In preferredaspects of the present disclosure, the fluorescent compound iscovalently encapsulated within the core of the nanoparticle.

In some aspects, fluorescent compound can be, but is not limited to, anear infrared fluorescent (NIRF) dye that is positioned within thesilica core of the nanoparticle, that can provide greater brightness andfluorescent quantum yield relative to the free fluorescent dye. It iswell-known that the near infrared-emitting probes exhibit decreasedtissue attenuation and autofluorescence (Burns et al. “Fluorescentsilica nanoparticles with efficient urinary excretion for nanomedicine”,Nano Letters (2009) 9(1):442-448).

Fluorescent compounds that may be used (e.g., encapsulated by an NDC) inthe present disclosure, include, but are not limited to, Cy5, Cy5.5(also known as Cy5++), Cy2, fluorescein isothiocyanate (FITC),tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7,fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red(sulforhodamine 101 acid chloride), LIGHTCYCLER®-Red 640,LIGHTCYCLER®-Red 705, tetramethylrhodamine (TMR), rhodamine, rhodaminederivative (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G), therhodamine derivative JA133, Alexa Fluorescent Dyes (such as ALEXA FLUOR®488, ALEXA FLUOR® 546, ALEXA FLUOR® 633, ALEXA FLUOR® 555, and ALEXAFLUOR® 647), 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide,aminomethylcoumarin (AMCA), Spectrum Green, Spectrum Orange, SpectrumAqua, LISSAMINE™, and fluorescent transition metal complexes, such aseuropium.

Fluorescent compounds that can be used also include fluorescentproteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP),blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, azurite,mKalamal), cyan fluorescent protein and derivatives (CFP, ECFP,Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP,Citrine, Venus, YPet) (WO 2008/142571, WO 2009/056282, WO 1999/22026).

In preferred aspects of the present disclosure, the fluorescent compoundis selected from the group consisting of Cy5 and Cy5.5. In preferredaspects, the fluorescent compound is Cy5.

A fluorescent nanoparticle may be synthesized by the steps of: (1)covalently conjugating a fluorescent compound, such as a reactivefluorescent dye (e.g., Cy5), with a reactive moiety including, but notlimited to, maleimide, iodoacetamide, thiosulfate, amine,N-hydroxysuccimide ester, 4-sulfo-2,3,5,6-tetrafluorophenyl(STP) ester,sulfosuccinimidyl ester, sulfodichlorophenol esters, sulfonyl chloride,hydroxyl, isothiocyanate, carboxyl, to an organo-silane compound, suchas a co-reactive organo-silane compound, to form a fluorescent silicaprecursor, and reacting the fluorescent silica precursor to form afluorescent core; or (2) reacting the fluorescent silica precursor witha silica forming compound, such as tetraalkoxysilane, to form afluorescent core. The fluorescent core may then be reacted with a silicaforming compound, such as a tetraalkoxysilane, to form a silica shell onthe core, to provide the fluorescent nanoparticle.

Fluorescent silica-based nanoparticles are known in the art and aredescribed by U.S. Pat. Nos. 8,298,677 B2, 9,625,456 B2, 1,0548,997 B2,9,999,694 B2, 1,0039,847 B2 and 10,548,998 B2, the contents of which areeach incorporated herein by reference in their entireties.

In preferred aspects of the present disclosure, the NDCs comprise ananoparticle that comprises a silica-based core and a silica shellsurrounding at least a portion of the core and polyethylene glycol (PEG)is covalently bonded to the surface of the nanoparticle, and afluorescent compound is covalently encapsulated within the core of thenanoparticle.

Targeting Ligand

The NDCs of the present disclosure may comprise a targeting ligand thatis attached to the nanoparticle directly or indirectly through a spacergroup. NDCs with targeting ligands can enhance internalization of thepayload/drugs in tumor cells and/or deliver drugs into tumor cells dueto increased permeability, as well as the targeting ability of the NDC.The targeting ligand can allow the nanoparticle to target a specificcell type through the specific binding between the ligand and thecellular component. The targeting ligand may also facilitate entry ofthe nanoparticle into the cell or barrier transport, for example, forassaying the intracellular environment.

The targeting ligands of the present disclosure are capable of bindingto receptors on tumor cells. Specifically, the targeting ligands canbind to the folate receptor (FR), including all four human isoforms ofFR, including FR alpha (FRα, also known as FOLR1), FR beta (FRβ, alsoknown as FOLR2), FR gamma (FRγ, also known as FOLR3), and FR delta (FRδ,also known as FOLR4). Conjugation of FR targeting ligand to the surfaceof the nanoparticle of the present disclosure allows for targetedtherapy of FR-overexpressing cancerous cells, tissues, and tumors. Forexample, NDCs of the present disclosure comprising targeting ligandsthat can bind to folate receptor alpha (FRα), such as folic acid, may beused for targeting ovarian cancer, endometrial cancer, fallopian tubecancer, peritoneal cancer, cervical cancer, breast cancer, lung cancer,mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophagealcancer, colon cancer, rectal cancer, and stomach cancer), pancreaticcancer, bladder cancer, kidney cancer, liver cancer, head and neckcancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, andtesticular cancer, acute myeloid leukemia (AML, e.g., pediatric AML).NDCs of the present disclosure comprising targeting ligands that canbind to folate receptor beta (FRβ) may be used for targeting acutemyeloid leukemia (AML, e.g., pediatric AML), chronic myelogenousleukemia (CML), and tumor associated macrophages. Tumor associatedmacrophages can be targeted as a means to modify the immune status ofthe tumor. Without wishing to be bound by theory, the binding affinityof FR-targeted NDCs to folate receptors can be enhanced due tomultivalence effect.

Folate receptor can be highly expressed in solid tumor cells, includingovarian, kidney, lung, brain, endometrial, colorectal, pancreatic,gastric, prostate, breast and non-small-cell lung cancers. FR isover-expressed in other cancers including fallopian tube cancer,cervical cancer, mesothelioma, uterine cancer, esophageal cancer,stomach cancer, bladder cancer, liver cancer, head and neck cancer,thyroid cancer, skin cancer, and testicular cancer. FR is alsoover-expressed in hematological malignancies, such as acute myeloidleukemia (AML) and chronic myelogenous leukemia (CML).

In preferred aspects of the present disclosure, the targeting ligandsbind to folate receptor alpha (FRa), folate receptor beta (FRβ), orboth.

The present disclosure provides FR-targeting ligands that are capable ofbinding to specific cell types having elevated levels of FRa, such as,but not limited to, cancer (e.g., adenocarcinomas) of uterus, ovary,breast, cervix, kidney, colon, testicles (e.g., testicularchoriocarcinoma), brain (e.g., ependymal brain tumors), malignantpleural mesothelioma, and nonfunctioning pituitary adenocarcinoma. Thepresent disclosure also provides FR-targeting ligands that are capableof targeting acute myeloid leukemia (AML, e.g., pediatric AML), chronicmyelogenous leukemia (CML), and tumor associated macrophages. Thetargeting ligand can be any suitable molecule that can bind a FR, suchas FRa, such as a small organic molecule (e.g., folate or a folateanalog), an antigen-binding portion of an antibody (e.g. a Fab fragment,a Fab′ fragment, a F(ab′)2 fragment, a scFv fragment, a Fv fragment, adsFv diabody, a dAb fragment, a Fd′ fragment, a Fd fragment, or anisolated complementarity determining region (CDR) region), an antibodymimetic (e.g., aptamer, an affibody, affilin, affimer, anticalin,avimer, Darpin, and the like), a nucleic acid, lipid, and the like.

In aspects of the present disclosure, the targeting ligand is folicacid, or a folate receptor binding derivative thereof. It will beunderstood that “folic acid” can encompass any amide or ester derivativeof folic acid. For example, free folic acid may be modified to beconjugated to the nanoparticle via a spacer group, such as PEG or a PEGderivative (e.g., by forming an amide bond between the terminalcarboxylic acid of folic acid, and a nitrogen atom of the spacer group).

The FR-targeted NDCs may not only accumulate in a cancer cell or tumor,but may also penetrate the tumor tissue and deliver payloads to theentire tumor tissue for optimal treatment efficacy. Without wishing tobe bound by any particular theory or mechanism, it is believed that thetargeting ligands bind to the specific receptor groups on the surface ofthe cancer cell, resulting in receptor-mediated cell uptake of NDCs.This receptor-mediated cell uptake of NDCs happens via the endocytosisprocess, and eventually traffics NDCs to endosomes and lysosomes incancer cells.

In aspects of the present disclosure, the NDCs comprise a targetingligand that is attached to the nanoparticle directly or indirectlythrough a spacer group. For example, the targeting ligand can beattached to the nanoparticle directly via the silica of the nanoparticle(i.e., covalently bonded). In preferred aspects, the targeting ligand isattached to the nanoparticle indirectly through a suitable spacer group.

The spacer group can be any group that can act as a spacer, e.g., as aspacer between a targeting ligand and the nanoparticle, and attach thetargeting ligand to the nanoparticle. The spacer group may be a divalentlinker, such as a divalent linker that comprises a chain length ofbetween about 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, ora combination thereof), such as between about 5 and about 100 atoms,between about 5 and about 80 atoms, between about 10 and about 80 atoms,between about 10 and about 70 atoms, between about 10 and about 30atoms, between about 20 and about 30 atoms, between about 30 and about80 atoms, or between about 30 and about 60 atoms. Suitable spacer groupsmay comprise an alkylene, alkenylene, alkynylene, heteroalkylene (e.g.,PEG), carbocyclyl, heterocyclyl, aryl, heteroaryl, or a combinationthereof. For example, the spacer group may comprise a PEG group, analkylene group, or a combination thereof. The spacer group may besubstituted or unsubstituted, e.g., the spacer group may comprise asubstituted alkylene, substituted heteroalkylene, or a combinationthereof. For example, the spacer group may comprise a PEG group (or PEGspacer), an alkylene group (or alkylene spacer), one or moreheteroatoms, and/or one or more cyclic groups (e.g., heterocyclylenegroups, such as a piperazine).

The targeting ligand, such as folic acid, may be attached to thenanoparticle indirectly through a PEG spacer group. The folic acid maybe present in the NDC as an amide, e.g., to facilitate conjugation to aPEG spacer group or other divalent linker, e.g., as shown in FIG. 1 .The number of PEG monomers in a PEG spacer may range from 2 to 20, from2 to 10, from 2 to 8, or from 2 to 5. In preferred aspects, the numberof PEG groups as spacers in a functionalized FR-targeting ligand is 3.

The average nanoparticle to targeting ligand (e.g., folic acid) ratiomay range from about 1 to about 50, from about 1 to about 40, from about1 to about 30, or from about 1 to about 20. For example, the averagenanoparticle to targeting ligand (e.g., folic acid) ratio may be about1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25,1:26, 1:27, 1:28, 1:29, 1:30, 1:40, or 1:50. For example, the averagenanoparticle to targeting ligand ratio may range from about 1 to about20, e.g., the average number of folic acid molecules on eachnanoparticle may be between about 5 and about 10, between about 10 andabout 15, or between about 15 and about 20, e.g., about 1, about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, or about 15 folic acid moleculesper nanoparticle. An NDC disclosed herein may comprise about 10 folicacid molecules. An NDC disclosed herein may comprise about 11 folic acidmolecules. An NDC disclosed herein may comprise about 12 folic acidmolecules. An NDC disclosed herein may comprise about 13 folic acidmolecules. An NDC disclosed herein may comprise about 14 folic acidmolecules. An NDC disclosed herein may comprise about 15 folic acidmolecules.

A smaller number of targeting ligands attached to the nanoparticle mayhelp maintain the hydrodynamic diameter of the nanoparticle, e.g., tomeet the renal clearance cutoff size range (Hilderbrand et al.,Near-infrared fluorescence: Application to in vivo molecular imaging,Curr. Opin. Chem. Biol., (2010) 14:71-79). The number of targetingligands measured may be an average number of targeting ligands attachedto more than one nanoparticle. Alternatively, one nanoparticle may bemeasured to determine the number of targeting ligands attached.

The number of targeting ligands attached to the nanoparticle can bemeasured by any suitable methods, such as, but not limited to, opticalimaging, fluorescence correlation spectroscopy (FCS), UV-Vis,chromatography, mass spectroscopy, or indirect enzymatic analysis.

The targeting ligand can be attached to the nanoparticle via covalentbonding to the silica of the nanoparticle (e.g., indirectly through aspacer group). The ligand may be conjugated to a nanoparticle (e.g., viaa functional group on the nanoparticle surface) described herein, forexample, using coupling reactions, Click Chemistry (e.g., a 3+2 ClickChemistry reaction), cycloaddition (e.g., a 3+2 or 2+4 cycloadditionreaction, using the appropriate functional groups), or conjugation via acarboxylate, ester, alcohol, carbamide, aldehyde, amine, sulfur oxide,nitrile oxide, nitrone, nitrogen oxide, halide, or any other suitablecompound known in the art.

In preferred aspects of the present disclosure, the conjugation ofFR-targeting ligands can be accomplished by “click chemistry” reactionusing a diarylcyclooctyne (DBCO) group. Any suitable reaction mechanismmay be adapted in the present disclosure for “click chemistry”, so longas facile and controlled attachment of the targeting ligand to thenanoparticle can be achieved.

In some aspects, a triple bond (e.g., alkyne, e.g., terminal alkyne) isintroduced onto the surface of a nanoparticle (e.g., via a PEGcovalently conjugated with the shell of the nanoparticle, or throughanother suitable linker or spacer group). Separately, an azide bond, orother group that is reactive with a triple bond, may be introduced ontothe desired targeting ligand. For example, folic acid may be modified byconjugating the terminal carboxylic acid of folic acid with a spacergroup (e.g., a PEG moiety), that comprises an azide at one terminus).The nanoparticle (e.g., PEGylated nanoparticle) comprising the freetriple bond, and the targeting ligand (comprising a group reactive withthe triple bond), can be mixed (with or without a copper or other metalcatalyst) to effect cycloaddition of the group reactive with the triplebond (e.g., azide) to the triple bond, resulting in the conjugation ofthe targeting ligand with the nanoparticle (e.g., “Click Chemistry”).Many variations of this approach can also be used, as will be readilyapparent to a person of ordinary skill in the art.

An azide functionalized FR-ligand (where the FR-ligand may comprise aspacer group, and the spacer group may possess the azide group) can beattached to the nanoparticle either directly or indirectly via an alkyne(e.g., DBCO group). Spacer groups, such as, but not limited to PEGgroups, can be present in a FR-targeting ligand precursor, and maypossess a terminal group (e.g., azide) to facilitate conjugation to thenanoparticle, and after conjugation, the spacer group may be disposedbetween the targeting ligand and the nanoparticle. For example, theFR-targeting ligand precursor may comprise a structure of Formula (D-1):

wherein y is an integer of 0 to 20 (e.g., 3). For example, y may be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20,e.g., 2, 3, or 4.

In some aspects, the FR-targeting ligand may be functionalized with asuitable terminal group, such as, but not limited to an azide group. Theazide functionalized FR-ligand can be attached to the nanoparticleeither directly or indirectly via the DBCO groups. Spacer groups, suchas, but not limited to PEG groups can be present between the azidefunctionalized FR-ligand and the nanoparticle. In preferred aspects, theFR-targeting ligand is functionalized to include spacer groups, such as,but not limited to PEG groups that terminate with an azide group thatreacts with the DBCO groups on the surface of the nanoparticle.

The functionalization of FR-targeting ligand may include hydrophilic PEGgroups as spacers, that may enhance solubility in water, and may reduceor eliminate aggregation and precipitation of the nanoparticle.

In aspects of the present disclosure, the number of PEG groups asspacers that can be present in a functionalized FR-targeting ligand maybe in the range of from 2 to 20, from 2 to 10, from 2 to 8, or from 2 to5. In preferred aspects, the number of PEG groups as spacers in afunctionalized FR-targeting ligand is 3.

The NDCs of the present disclosure comprising a targeting ligand maycomprise a structure of Formula (NP-2):

wherein x is an integer of 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, e.g., 4), and y is an integer of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 3), and thesilicon atom is a part of the nanoparticle (e.g., bonded with the silicashell of a core-shell silica nanoparticle). For example, x may be 4, andy may be 3. Each nanoparticle of the NDCs disclosed herein may comprisemore than one molecule of Formula (NP-2), for example, the nanoparticlemay comprise between about 1 and about 20 molecules of Formula (NP-2),e.g., between about 5 and about 20 molecules of Formula (NP-2), betweenabout 8 and about 15 molecules of Formula (NP-2), between about 10 andabout 15 molecules of Formula (NP-2), e.g., about 1, about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, about18, about 19, or about 20 molecules of Formula (NP-2). An NDC disclosedherein may comprise about 12 molecules of Formula (NP-2). An NDCdisclosed herein may comprise about 13 molecules of Formula (NP-2).

Linker-Payload Conjugate

The NDCs of the present disclosure can also comprise a linker-payloadconjugate that is attached to the nanoparticle directly or indirectlythrough a spacer group. In preferred aspects, the linker-payloadconjugate is attached to the nanoparticle through a spacer group. Thepayload may be exatecan, or a salt or analog thereof

The spacer group can be any group that can act as a spacer, e.g., as aspacer between a payload/linker conjugate and the nanoparticle, andattach the linker-payload conjugate to the nanoparticle. The spacergroup may be a divalent linker, such as a divalent linker that comprisesa chain length of between about 5 and about 200 atoms (e.g., carbonatoms, heteroatoms, or a combination thereof), such as between about 5and about 100 atoms, between about 5 and about 80 atoms, between about10 and about 80 atoms, between about 10 and about 70 atoms, betweenabout 10 and about 30 atoms, between about 20 and about 30 atoms,between about 30 and about 80 atoms, or between about 30 and about 60atoms. Suitable spacer groups may comprise an alkylene, alkenylene,alkynylene, heteroalkylene (e.g., PEG), carbocyclyl, heterocyclyl, aryl,heteroaryl, or a combination thereof. For example, the spacer group maycomprise a PEG group, an alkylene group, or a combination thereof. Thespacer group may be substituted or unsubstituted, e.g., the spacer groupmay comprise a substituted alkylene, substituted heteroalkylene, or acombination thereof. For example, the spacer group may comprise a PEGgroup (or PEG spacer), an alkylene group (or alkylene spacer), one ormore heteroatoms, and/or one or more or cyclic groups.

It will be understood that chemical modifications may be made to thepayload in order to make reactions of the payload with linker moreconvenient for purposes of preparing conjugates of the presentdisclosure. For example, a functional group, e.g., amine, hydroxyl, orsulfhydryl, may be appended to the payload (e.g., exatecan) at aposition which has minimal or an acceptable effect on the activity orother properties of the payload (e.g., exatecan). Alternatively, anexisting functional group on the payload (e.g., pendant amine group) maybe the point of attachment to the linker. For example, exatecan containsan amine functional group suitable for coupling to the linker moiety

The payload (e.g., exatecan payload, or a salt or analog thereof) can becleaved from the nanoparticle inside a cell, or a cell organelle, e.g.,by an enzyme, thereby releasing exatecan, e.g., inside the cell or cellorganelle). Exatecan is a topoisomerase 1 (Topo-1) inhibitor that canstabilize the complexes of DNA and Topo-1 enzyme, preventing DNArelegation and inducing lethal DNA strand breaks. The generation ofthese DNA lesions is effective for killing cancer cells, allowing NDCsof the present disclosure to achieve the desired therapeutic effect.

In preferred aspects of the present disclosure, the payload is exatecan,or a salt thereof. In other preferred aspects of the present disclosure,the payload is an analog of exatecan, or a salt thereof

In aspects of the present disclosure, the average nanoparticle topayload ratio ranges from 1 to 80, from 1 to 70, from 1 to 60, from 1 to50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 15, from 1 to 12and preferably from 1 to 10. For example, the average nanoparticle topayload (e.g., exatecan, or a salt or analog thereof) ratio may be about1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13,1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25,1:26, 1:27, 1:28, 1:29, 1:30, 1:32, 1:34, 1:36, 1:38, 1:40, 1:45, 1:50,1:55, 1:60, 1:65, 1:70, 1:75, or 1:80. For example, the average numberof exatecan molecules on each nanoparticle may be between about 5 andabout 10, between about 10 and about 15, between about 15 and about 20,between about 20 and about 25, or between about 25 and about 30, e.g.,about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24, about 25, about 26, about 27, about 28, about 29, orabout 30 exatecan molecules per nanoparticle. An NDC disclosed hereinmay comprise about 18 exatecan molecules. An NDC disclosed herein maycomprise about 19 exatecan molecules. An NDC disclosed herein maycomprise about 20 exatecan molecules. An NDC disclosed herein maycomprise about 21 exatecan molecules. An NDC disclosed herein maycomprise about 22 exatecan molecules. An NDC disclosed herein maycomprise about 23 exatecan molecules. An NDC disclosed herein maycomprise about 24 exatecan molecules. An NDC disclosed herein maycomprise about 25 exatecan molecules. An NDC disclosed herein maycomprise about 26 exatecan molecules. An NDC disclosed herein maycomprise about 27 exatecan molecules.

Vintafolide, developed by Endocyte and Merck & Co. is a small moleculedrug conjugate consisting of a small molecule targeting the FolateReceptor, which is over expressed on certain cancers, such as ovariancancer, and a chemotherapy drug, Vinblastine (U.S. Pat. Nos. 7,601,332B2 and 1,002,942 B2). However, vintafolide is capable of carrying singlemolecule of payload only, attached to the targeting moiety by apH-cleavable linker. In contrast to that, in the present disclosureseveral cytotoxic payloads (e.g., exatecan molecules) can beincorporated onto the surface of single nanoparticle.

The linkers in the linker-payload conjugates can be self-immolativelinkers that are capable of releasing the active payload in vitro aswell as in vivo under conditions sufficient for enzymatic release of theactive payload (e.g., a condition presenting an enzyme capable ofcatalyzing the release).

The linkers described herein can be used, for example, to attach acytotoxic drug payload (e.g., exatecan) to a carrier and/or a targetingmoiety (e.g., nanoparticle) that binds to a cancer cell (e.g., binds toa receptor on the surface of a cancer cell) and gets internalized intothe cell (e.g., through the endosome and lysosomal compartment). Onceinternalized, the linkers can be cleaved or degraded to release activecytotoxic drug. Specifically, the protease-cleavable linkers can releasetheir payload under the action of proteases such as cathepsin, trypsinor other proteases in the lysosomal compartment of the cell.

The cleavable linkers described herein may comprise a structure ofFormula (F):

wherein each instance of [AA] is a natural or non-natural amino acidresidue; z is an integer of 1 to 5; w is an integer of 1 to 4 (e.g., 2or 3); and each

denotes a point of attachment, e.g., to a spacer group (e.g., PEG) oranother portion of the linker, or to an exatecan molecule. For example,-[AA]_(w)- may comprise Val-Lys, Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys,Val-Arg, or Val-Ala, and z may be 2, wherein one

denotes an attachment to the oxygen atom of a PEG group, and the other

denotes an attachment to the nitrogen atom of exatecan. For example,-[AA]_(w)- may comprise Val-Lys.

The cleavable linkers described herein may comprise a structure ofFormula (F-1):

wherein one

denotes a point of attachment to the oxygen atom of a PEG group, and theother

denotes a point of attachment to the nitrogen atom of exatecan.

The linkers of this disclosure can be prepared from linker precursorsthat contain reactive groups at one or both ends of the molecule. Thereactive groups can be selected to allow conjugation to exatecan or ananalog thereof at one end, and also facilitate conjugation to thenanoparticle at the other end. It is desirable for the payload tocontain an amine, a hydroxyl, hydrazone, hydrazide or a sulfhydryl groupin order to facilitate conjugation to the linker. For example, exatecancomprises a primary amine group that can facilitate its conjugation tothe linker.

The linker-payload conjugate precursors can be attached to thenanoparticle using any suitable techniques and methods, and many suchtechniques are well-known in the art. See, e.g., WO 2017/189961, WO2015/183882, WO 2013/192609, WO 2016/179260 and WO 2018/213851, each ofwhich are hereby incorporated by reference in their entireties, whichdescribe silica-based core-shell or silica-based core nanoparticles thatcan be used to prepare targeted nanoparticle-based drug deliverysystems. Additionally, linker-payload conjugate precursors, orligand-linker precursors, can be attached to a nanoparticle using areaction or method described in Kolb et al. Angew. Chem. Int. Ed. (2001)40:2004-2021, which is incorporated herein by reference in its entirety.

The linker-payload conjugate may be attached to the nanoparticledirectly or indirectly through a spacer group, such as a spacer groupdescribed herein. Suitable spacer groups include, but are not limitedto, a divalent linker (e.g., a divalent linker described herein), suchas PEG spacer, or an alkylene spacer (e.g., a methylene spacer), whichmay further comprise a heteroatom or cyclic group (e.g., heterocyclylenegroup). The linker-payload conjugate can be absorbed into theinterstices or pores of the silica shell, or coated onto the silicashell of the nanoparticle, such as a fluorescent nanoparticle (e.g.,covalently attached to the surface of the nanoparticle). In otheraspects, where the silica shell is not covering all of the surface ofthe nanoparticle, the linker-payload conjugate can be associated withthe fluorescent core, such as by physical absorption or by bondinginteraction.

In some aspects, the linker-payload conjugate may also be associatedwith the PEG groups that are covalently bonded to the surface of thenanoparticle. For example, the linker-payload conjugate may be attachedto the nanoparticle through the PEG. The PEGs can have multiplefunctional groups for attachment to the nanoparticle and to thelinker-payload conjugate.

In specific aspects of the present disclosure, the linker-payloadconjugates (or linker-payload conjugate precursor) may be functionalizedwith a hydrophilic PEG spacer. The linker-payload conjugate precursormay be functionalized with a hydrophilic PEG spacer and/or suitableterminal group such as, but not limited to, an azide group, tofacilitate covalently attaching the linker-payload conjugate (e.g., viathe spacer group) to the surface of the nanoparticle, e.g., via reactionwith a DBCO group on the nanoparticle surface). Other terminal groupscan include a nitrile oxide or nitrone, e.g., for conjugation via a 3+2cycloaddition reaction, to a suitable group on the nanoparticle (e.g., adiene moiety).

The number of PEG groups as spacers that can be present in afunctionalized linker-payload conjugate (or precursor thereof) may rangefrom 0 to 20, e.g., from 2 to 20, from 2 to 10, or from 5 to 8, e.g., 5,6, 7, 8, 9, 10, 11, or 12. In preferred aspects, the number of PEGgroups as spacers in a functionalized linker-payload conjugate is 9.

For example, exatecan can be conjugated to a protease-cleavable linkerto form the linker-payload conjugate. This linker-conjugate can beprepared from a precursor functionalized with a PEG spacer that has aterminal reactive group, such as an azide, for further conjugation tothe surface of the nanoparticle, e.g., via a DBCO group.

The protease-cleavable linker can be designed to be labile to cathepsinB (Cat-B), an enzyme that is over-expressed in malignant tumors, therebyeffecting release of the cytotoxic agent, such as exatecan by aself-immolative process.

The linker payload conjugate precursor can comprise a structure ofFormula (E-1):

wherein y is an integer of 0 to 20, e.g., 5 to 15, e.g., 9.

The linker and linker-payload conjugates described in the presentdisclosure have several advantages, ranging from superior serumstability to faster release kinetics mechanism, relative to conventionaldrug delivery platforms, linkers, or linker-payload conjugates. Also,the ability to pair these linkers with a variety of chemical groupsprovides the opportunity for the selective release of freepayload/drugs, with minimal derivatization, that is a significantadvantage.

In preferred aspects of the present disclosure, the linker in thelinker-payload conjugate is a protease-cleavable linker.

The NDCs of the present disclosure comprising a payload-linker moietymay comprise a structure of Formula (NP-3):

wherein xis an integer of 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10, e.g., 4), and y is an integer of 0 to 20 (e.g., 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 9), andthe silicon atom is a part of the nanoparticle (e.g., bonded with thesilica shell of a core-shell silica nanoparticle. For example x may be4, and y may be 9. An NDC disclosed herein may comprise more than onemolecule of Formula (NP-3), for example, the nanoparticle may comprisebetween about 1 and about 80 molecules of Formula (NP-3), e.g., betweenabout 1 and about 60 molecules of Formula (NP-3), between about 1 andabout 40 molecules of Formula (NP-3), between about 1 and about 30molecules of Formula (NP-3), between about 10 and about 30 molecules ofFormula (NP-3), between about 15 and about 25 molecules of Formula(NP-3), e.g., about 1, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19, about 20, about21, about 22, about 23, about 24, about 25, about 26, about 27, about28, about 29, or about 30 molecules of Formula (NP-3).

Upon contact with a protease (e.g., within a cancer cell, such as withinthe lysosome of a cancer cell), an NDC of the present disclosure mayundergo cleavage to release free exatecan. The cleavage of an NDCdisclosed herein may concomitantly release exatecan, carbon dioxide, and4-aminobenzyl alcohol from the NDC. For example, the cleavage of anexemplary NDC disclosed herein is provided in Scheme 1 below.

The NDCs disclosed herein may comprise both a molecule of Formula(NP-2), and a molecule of Formula (NP-3), e.g., each NDC may compriseabout 1 and about 20 molecules of Formula (NP-2), and about 1 and about30 molecules of Formula (NP-3). For example, each NDC may comprise about10 and about 15 molecules of Formula (NP-2), and about 15 and about 25molecules of Formula (NP-3). An NDC disclosed herein may comprise anaverage of 13 molecules of Formula (NP-2), and an average of 21molecules of Formula (NP-3); an average of 12 molecules of Formula(NP-2), and an average of 25 molecules of Formula (NP-3); an average of12 molecules of Formula (NP-2), and an average of 20 molecules ofFormula (NP-3).

This disclosure provides compositions and methods directed to ananoparticle-drug conjugate (NDC) comprising: a nanoparticle; atargeting ligand that binds to folate receptor; and a linker-payloadconjugate, wherein the NDC has an average diameter between about 1 nmand about 10 nm. For example, a nanoparticle comprising folic acid as atargeting ligand, and a linker-payload conjugate comprising exatecanconjugated via a protease-cleavable linker, wherein the NDC has anaverage diameter between about 1 nm and about 10 nm.

FIG. 1 illustrates a representative nanoparticle-drug conjugate (NDC)that has an average diameter of about 6 nm, comprising a nanoparticlethat comprises a silica-based core and a silica shell surrounding atleast a portion of the core, polyethylene glycol (PEG) covalently bondedto the surface of the nanoparticle, and a fluorescent compound (Cy5)covalently encapsulated within the core of the nanoparticle, folic acid(FA) as the targeting ligand that can bind to a folate receptor, and alinker-payload conjugate that comprises a protease-cleavablelinker-exatecan conjugate. It will be understood that “folic acid” isintended to encompass any amide or ester derivative of folic acid, e.g.,as shown in FIG. 1 where folic acid is covalently attached to the spacergroup (PEG) via an amide group.

The NDC may have an average diameter between about 5 nm to about 8 nm,or between about 6 nm to about 7 nm. The average diameter of NDCs can bemeasured by any suitable methods, such as, but not limited to,fluorescence correlation spectroscopy (FCS) (see, e.g., FIG. 6 ) and gelpermeation chromatography (GPC) (FIG. 7 ).

The NDCs of the present disclosure can comprise nanoparticles that canbe functionalized with contrast agents for positron emission tomography(PET), single photon emission computed tomography (SPECT), computerizedtomography (CT), magnetic resonance imaging (MRI), and optical imaging(such as fluorescence imaging including near-infrared fluorescence(NIRF) imaging, bio luminescence imaging, or combinations thereof).

A contrast agent, such as a radionuclide (radiolabel) including, but notlimited to ⁸⁹Zr, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ¹²⁴I and ¹⁷⁷Lu, may be attached to thenanoparticle. Alternatively, the nanoparticle can be attached to achelator moiety, for example, DFO, DOTA, TETA and DTPA, that is adaptedto bind a radionuclide. Such nanoparticle may be detected by PET, SPECT,CT, MRI, or optical imaging (such as fluorescence imaging includingnear-infrared fluorescence (NIRF) imaging, bio luminescence imaging, orcombinations thereof).

The radionuclide can additionally serve as a therapeutic agent forcreating a multitherapeutic platform. This coupling allows thetherapeutic agent to be delivered to the specific cell type through thespecific binding between the targeting ligand and the cellularcomponent.

Protease-Cleavable Linker-Payload Conjugates

A linker-payload conjugate may comprise a compound of Formula (I)

or a salt thereof, wherein,

line represents an attachment to the nanoparticle through a spacergroup; A is a dipeptide selected from the group consisting of Val-Cit,Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala, or A is atetrapeptide selected from the group consisting of Val-Phe-Gly-Sar,Val-Cit-Gly-Sar, Val-Lys-Gly-Sar, Val-Ala-Gly-Sar, Val-Phe-Gly-Pro,Val-Cit-Gly-Pro, Val-Lys-Gly-Pro, Val-Ala-Gly-Pro, Val-Cit-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid,Val-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid, Val-Phe-Gly-any natural or unnatural N-alkyl substituted alphaamino acid, Val-Ala-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Phe-Lys-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, and Trp-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid; Payload is exatecan, and theprimary amine group of exatecan is represented by Z; R¹ and R² in eachoccurrence is independently hydrogen, substituted or unsubstituted C₁₋₆alkyl or substituted or unsubstituted C₁₋₆ alkoxy, or hydroxyl; R³ andR⁴ in each occurrence is independently hydrogen, halo, substituted orunsubstituted C₁₋₆ alkyl, or substituted or unsubstituted C₁₋₆ alkoxy;R⁵ is selected from the group consisting of hydrogen, substituted orunsubstituted C₁₋₆ alkyl; substituted or unsubstituted C₃₋₇ cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted C₅₋₆ heterocycloalkyl; withthe proviso that, when A is a dipeptide, R⁵ is H; R^(a), R^(b), andR^(c) in each occurrence is independently hydrogen or substituted orunsubstituted C₁₋₆ alkyl; X is absent, —O—, —CO— or —NR^(a)—; Y isabsent,

wherein the carbonyl in

is bonded to Z;

-   with the proviso that, when Y is

X is absent and n is 1; when Y is

X is absent and n is 0; when Y is

X is absent and n is 0; and/or when X is —CO—, Y is absent and n is 0;X₁ and X₂ are independently —CH— or —N—; X₃ is —CH—; X₄ is —CH—; Z is—NRC— or —O—; n is 0 or 1; q is 1 to 3.

In preferred aspects of Formula (I), A is Val-Lys; R¹-R⁵ are eachindependently hydrogen; X is absent; Y is

wherein the carbonyl in

is bonded to Z; n is 1; X₁, X₂, X₃, and X₄ are each independently —CH—;Z is —NR^(c)— wherein R^(c) is hydrogen, and wherein the N is thenitrogen atom present in the exatecan payload.

In the linker-payload conjugate of Formula (I), the payload may beexatecan, which has a functional group that is bonded to the linker,wherein the functional group is an amine (when exatecan is bonded to thelinker, it is a secondary amine, and once released (or prior toconjugation), i.e., as a separate molecular entity, the amine ofexatecan is a primary amine).

Exemplary Linker payload Conjugates: Representative linker-payloadconjugates of the present disclosure include, but are not limited to thefollowing sub-structures, wherein

line represents a direct bond to the nanoparticle or an indirect bond tothe nanoparticle through a spacer group. Suitable spacer groups include,but are not limited to a PEG spacer, or an alkylene spacer (e.g.,methylene spacer), which may further comprise heteroatoms, or cyclicgroups (e.g., heterocyclylene groups). In preferred aspects, the spacergroup is a PEG spacer.

An exemplary linker-payload conjugate of Formula (I) of the presentdisclosure includes the following sub-structure:

Linkers and Precursors Thereof The linkers of this disclosure, and/ortheir precursors, can contain reactive groups at both ends of themolecule. The reactive groups can be selected to allow conjugation toexatecan or a salt or analog thereof at one end, and also facilitateconjugation to a nanoparticle (e.g., via a spacer group) at the otherend. For example, the linker can connect to exatecan via a chemicallyreactive functional group that is a part of the exatecan, such as theprimary amine of exatecan (that becomes a secondary amine uponconjugation to the linker).

The linker can be conjugated to a functionalized polyethylene glycol ora C₅-C₆ alkyl chain via a chemically reactive functional group that is apart of the linker such as a primary or secondary amine or carboxylgroup.

Protease-cleavable Linkers: Proteases are involved in all stages ofcancer disease from tumor cells growth and survival, to angiogenesis andinvasions. Therefore, they can be utilized to treat cancer as selectivetriggers towards activation of linker/payload system. This disclosurerelates to linkers that are cleavable by the action of proteases therebyreleasing the free payload (e.g., exatecan). Lysosomal proteases such ascathepsin B and serine proteases such as cathepsin A ortripeptidyl-peptidase I have been extensively studied in the context ofprodrug development. Proteolytic enzymes such as caspases are alsowell-known to be utilized as biological triggers for the selectiveactivation of payload or for specific cargo delivery to a target cellsuch as a cancer cell.

A linker (or precursor thereof) can comprise a compound of Formula (I-A)

wherein: A is a dipeptide selected from the group consisting of Val-Cit,Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala, or A is atetrapeptide selected from the group consisting of Val-Phe-Gly-Sar,Val-Cit-Gly-Sar, Val-Lys-Gly-Sar, Val-Ala-Gly-Sar, Val-Phe-Gly-Pro,Val-Cit-Gly-Pro, Val-Lys-Gly-Pro, Val-Ala-Gly-Pro, Val-Cit-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid,Val-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid, Val-Phe-Gly-any natural or unnatural N-alkyl substituted alphaamino acid, Val-Ala-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Phe-Lys-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, and Trp-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid; R¹ and R² in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ alkoxy, or hydroxy; R³ and R⁴ in eachoccurrence is independently hydrogen, halo, substituted or unsubstitutedC₁₋₆ alkyl or substituted or unsubstituted C₁₋₆ alkoxy; R⁵ is selectedfrom the group consisting of hydrogen, substituted or unsubstituted C₁₋₆alkyl; substituted or unsubstituted C₃₋₇ cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, andsubstituted or unsubstituted C₅₋₆ heterocycloalkyl, with the provisothat, when A is a dipeptide, R⁵ is H; R¹′, R²′, R³′, R⁴′ and R⁵′ in eachoccurrence is independently hydrogen, substituted or unsubstituted C₁₋₆alkyl or substituted or unsubstituted C₁₋₆ cycloalkyl; X is absent, —O—,—CO— or —NR^(a)—; Y is absent,

wherein the carbonyl in

is bonded to Z₁, with the proviso that, when Y is

X is absent and n is 1; with the proviso that, when Y is

X is absent and n is 0, with the proviso that, when Y is

X is absent and n is 0 or 1; with the proviso that , when X is —CO—, Yis absent and n is 0; X₃ is —CH—; X₄ is —CH—; Z₁ is a functional groupselected from the group consisting of halo, hydroxy, —OSO₂—CH₃,—OSO₂CF₃, 4-nitrophenoxy, —COCl, and —COOH; Z₂ is a functional groupselected from the group consisting of —NH₂, —NHR^(c), and —COOH; or Z₂is —C(O)—T₁; T₁ is a functionalized polyethylene glycol or a C₅-C₆ alkylchain that has a terminal group selected from the group consisting ofazide,

R^(a), R^(b) and R^(c) in each occurrence is independently hydrogen orsubstituted or unsubstituted C₁₋₆ alkyl; n is 0 or 1; and q is 1 to 3.In certain aspects of Formula (I-A), A is Val-Lys; R¹-R⁵ are eachindependently hydrogen; X is absent; Y is

wherein the carbonyl in

is bonded to Z; n is 1; X₁, X₂, X₃ and X₄ are each independently —CH—;Z₁ is a functional group selected from the group consisting of halo,hydroxy, —OSO₂—CH₃, —OSO₂CF₃, 4-nitrophenoxy, —COCl, and —COOH; Z₂ is afunctional group selected from the group consisting of —NH₂, —NHR^(c),and —COOH or Z₂ is —C(O)—T₁, wherein T₁ is as defined in Formula (I-A).

Pharmaceutical Compositions

The present disclosure further provides a pharmaceutical composition fortreating a disease (e.g., cancer, such as a cancer associated withfolate receptor expressing tumor), wherein the composition comprises aneffective amount of an NDC described herein.

In specific aspects of the present disclosure, the pharmaceuticalcomposition comprising the NDCs can be used to treat cancer selectedfrom the group consisting of ovarian cancer, endometrial cancer,fallopian tube cancer, cervical cancer, breast cancer, lung cancer,mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophagealcancer, colon cancer, rectal cancer, and stomach cancer), pancreaticcancer, bladder cancer, kidney cancer, liver cancer, head and neckcancer, brain cancer, thyroid cancer, skin cancer, prostate cancer,testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML),and chronic myelogenous leukemia (CML). The pharmaceutical compositioncomprising the NDCs may also be used for targeting tumor associatedmacrophages, e.g., to modify the immune status of a tumor in a subject.

The pharmaceutical compositions of the present disclosure may comprise apharmaceutically acceptable excipient, such as a non-toxic carrier,adjuvant, diluent, or vehicle that does not negatively impact thepharmacological activity of the NDCs with which it is formulated.Pharmaceutically acceptable excipients useful in the manufacture of thepharmaceutical compositions of the present disclosure are any of thosethat are well known in the art of pharmaceutical formulation, and caninclude inert diluents, dispersing and/or granulating agents, surfaceactive agents and/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils.Pharmaceutically acceptable excipients useful in the manufacture of thepharmaceutical compositions of the present disclosure include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins (e.g., human serum albumin), buffer substances (e.g.,phosphates), glycine, sorbic acid, potassium sorbate, glyceride mixtures(e.g., mixtures of saturated vegetable fatty acids), water, salts orelectrolytes (e.g., protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts), colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol, and wool fat.

The pharmaceutical compositions of the present disclosure may beadministered orally in the form of a suitable pharmaceutical unit dosageform. The pharmaceutical compositions of the present disclosure may beprepared in many forms that include tablets, hard or soft gelatincapsules, aqueous solutions, suspensions, liposomes, and otherslow-release formulations, such as shaped polymeric gels.

Suitable modes of administration for the NDCs or composition include,but are not limited to, oral, intravenous, rectal, sublingual, mucosal,nasal, ophthalmic, subcutaneous, intramuscular, transdermal, spinal,intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial,lymphatic administration, intra-tumoral, and other routes suitable forsystemic delivery of active ingredients.

The present pharmaceutical composition may be administered by any methodknown in the art, including, without limitation, transdermal (passivevia patch, gel, cream, ointment or iontophoretic); intravenous (bolus,infusion); subcutaneous (infusion, depot); transmucosal (buccal andsublingual, e.g., orodispersible tablets, wafers, film, and effervescentformulations); conjunctival (eyedrops); rectal (suppository, enema)); orintradermal (bolus, infusion, depot). The composition may be deliveredtopically.

Oral liquid pharmaceutical compositions may be in the form of, forexample, aqueous or oily suspensions, solutions, emulsions, syrups orelixirs, or may be presented as a dry product for constitution withwater or other suitable vehicle before use. Such liquid pharmaceuticalcompositions may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

The pharmaceutical compositions of the present disclosure may also beformulated for parenteral administration (e.g., by injection, forexample, bolus injection or continuous infusion) and may be presented inunit dosage form in ampules, pre-filled syringes, infusion containers(e.g., small volume infusion containers), or multi-dose containers, thatmay contain an added preservative.

The pharmaceutical compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulating agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the pharmaceutical compositions of the presentdisclosure may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

For topical administration (e.g., to the epidermis), the pharmaceuticalcompositions may be formulated as an ointment, cream, or lotion, or asthe active ingredient of a transdermal patch. Suitable transdermaldelivery systems are disclosed, for example, in A. Fisher et al. (U.S.Pat. No. 4,788,603), and R. Bawa et al. (U.S. Pat. Nos. 4,931,279;4,668,506; and 4,713,224), which are incorporated herein by reference intheir entireties. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase and will in general also contain one or more emulsifying agents,stabilizing agents, dispersing agents, suspending agents, thickeningagents, or coloring agents. The pharmaceutical compositions can also bedelivered via ionophoresis, e.g., as disclosed in U.S. Pat. Nos.4,140,122; 4,383,529; or 4,051,842, each of which are incorporatedherein by reference in their entireties.

Pharmaceutical compositions suitable for topical administration in themouth include unit dosage forms such as lozenges comprising apharmaceutical composition of the present disclosure in a flavored base,such as sucrose and acacia or tragacanth; pastilles comprising thepharmaceutical composition in an inert base such as gelatin and glycerinor sucrose and acacia; mucoadherent gels, and mouthwashes comprising thepharmaceutical composition in a suitable liquid carrier.

For topical administration to the eye, the pharmaceutical compositionscan be administered as drops, gels (S. Chrai et al, U.S. Pat. No.4,255,415), gums (S. L. Lin et al, U.S. Pat. No. 4,136,177) or via aprolonged-release ocular insert (A. S. Michaels, U.S. Pat. No. 3,867,519and H. M. Haddad et al., U.S. Pat. No. 3,870,791), each of which areincorporated herein by reference in their entireties.

When desired, the above-described pharmaceutical compositions can beadapted to give sustained release of a therapeutic compound employed,e.g., by combination with certain hydrophilic polymer matrices, e.g.,comprising natural gels, synthetic polymer gels or mixtures thereof.

Pharmaceutical compositions suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the pharmaceutical composition withthe softened or melted carrier(s) followed by chilling and shaping inmolds.

Pharmaceutical compositions suitable for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams, or sprayscontaining, in addition to the nanoparticles and the therapeutic agent,a carrier. Such carriers are well known in the art.

For administration by inhalation, the pharmaceutical compositionsaccording to the present disclosure are conveniently delivered from aninsufflator, nebulizer or a pressurized pack or other convenient meansof delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thepharmaceutical compositions of the present disclosure may take the formof a dry powder composition, for example, a powder mix of thepharmaceutical composition and a suitable powder base such as lactose orstarch. The powder composition may be presented in unit dosage form in,for example, capsules or cartridges or, e.g., gelatin or blister packsfrom which the powder may be administered with the aid of an inhalatoror insufflator.

For intra-nasal administration, the pharmaceutical compositions of thepresent disclosure may be administered via a liquid spray, such as via aplastic bottle atomizer. Typical of these are the MISTOMETER®(isoproterenol inhaler- Wintrop) and the MEDIHALER® (isoproterenolinhaler—Riker).

Pharmaceutical compositions of the present disclosure may also containother adjuvants such as flavorings, colorings, anti-microbial agents, orpreservatives.

It will be further appreciated that the amount of the pharmaceuticalcompositions suitable for use in treatment will vary not only with thetherapeutic agent selected but also with the route of administration,the nature of the condition being treated and the age and condition ofthe patient and will be ultimately at the discretion of the attendantphysician or clinician. For evaluations of these factors, see J. F.Brien et al., Europ. J. Clin. Pharmacol., 14, 133 (1978); andPhysicians' Desk Reference, Charles E. Baker, Jr., Pub., MedicalEconomics Co., Oradell, N.J. (41^(st) ed., 1987), each of which areincorporated herein by reference in their entireties.

Administration and Methods of Treatment

NDCs of the present disclosure can be administered to a subject. Thesubject can be a mammal, preferably a human. Mammals include, but arenot limited to, murines, rats, rabbits, simians, bovines, ovine, swine,canines, feline, farm animals, sport animals, pets, equine, andprimates.

NDCs may be administered to a subject by, but not restricted to, thefollowing routes: oral, intravenous, nasal, subcutaneous, local,intramuscular or transdermal. For example, the NDCs of the presentdisclosure may be administered to a subject intravenously.

The methods and compositions of the present disclosure can be used tohelp a physician or surgeon to identify and characterize areas ofdisease, such as cancers, including, but not restricted to, cancers thatoverexpress FR, to distinguish diseased and normal tissue, such asdetecting tumor margins that are difficult to detect using an ordinaryoperating microscope, e.g., in brain surgery, to help dictate atherapeutic or surgical intervention, e.g., by determining whether alesion is cancerous and should be removed or non-cancerous and leftalone, or in surgically staging a disease.

The methods and compositions of the present disclosure may be used, butare not limited to, metastatic disease detection, treatment responsemonitoring, and targeted delivery of payload, including by passing theblood-brain barrier.

The methods and compositions of the present disclosure can also be usedin the detection, characterization and/or determination of thelocalization of a disease, including early disease, the severity of adisease or a disease-associated condition, the staging of a disease,and/or monitoring a disease. The presence, absence, or level of anemitted signal can be indicative of a disease state.

The methods and compositions of the present disclosure can also be usedto monitor and/or guide various therapeutic interventions, such assurgical and catheter-based procedures, and monitoring drug therapy,including cell based therapies. The methods of the present disclosurecan also be used in prognosis of a disease or disease condition.Cellular subpopulations residing within or marginating the disease site,such as stem-like cells (“cancer stem cells”) and/orinflammatory/phagocytic cells may be identified and characterized usingthe methods and compositions of the present disclosure.

With respect to each of the foregoing, examples of such disease ordisease conditions that can be detected or monitored (before, during orafter therapy) include cancer (for example, melanoma, thyroid,colorectal, ovarian, lung, breast, prostate, cervical, skin, brain,gastrointestinal, mouth, kidney, esophageal, bone cancer), that can beused to identify subjects that have an increased susceptibility fordeveloping cancer and/or malignancies, i.e., they are predisposed todevelop cancer and/or malignancies, inflammation (for example,inflammatory conditions induced by the presence of cancerous lesions),cardiovascular disease (for example, atherosclerosis and inflammatoryconditions of blood vessels, ischemia, stroke, thrombosis), dermatologicdisease (for example, Kaposi's Sarcoma, psoriasis), ophthalmic disease(for example, macular degeneration, diabetic retinopathy), infectiousdisease (for example, bacterial, viral, fungal and parasitic infections,including Acquired Immunodeficiency Syndrome (AIDS)), immunologicdisease (for example, an autoimmune disorder, lymphoma, multiplesclerosis, rheumatoid arthritis, diabetes mellitus), central nervoussystem disease (for example, a neurodegenerative disease, such asParkinson's disease or Alzheimer's disease), inherited diseases,metabolic diseases, environmental diseases (for example, lead, mercuryand radioactive poisoning, skin cancer), bone-related disease (forexample, osteoporosis, primary and metastatic bone tumors,osteoarthritis) and a neurodegenerative disease.

The methods and compositions of the present disclosure, therefore, canbe used, for example, to determine the presence and/or localization oftumor and/or co-resident stem-like cells (“cancer stem cells”), thepresence and/or localization of inflammatory cells, including thepresence of activated macrophages, for instance in peritumoral regions,the presence and in localization of vascular disease including areas atrisk for acute occlusion (i.e., vulnerable plaques) in coronary andperipheral arteries, regions of expanding aneurysms, unstable plaque incarotid arteries, and ischemic areas. The methods and compositions ofthe present disclosure can also be used in identification and evaluationof cell death, injury, apoptosis, necrosis, hypoxia and angiogenesis(PCT/US2006/049222).

The methods of the present disclosure comprise administering to asubject in need thereof an effective amount of an NDC described herein.For example, the NDC can be administered to the subject in need thereofintravenously. An “effective amount” is an amount of the NDC thatelicits a desired biological or medicinal response under the conditionsof administration, such as an amount that reduces the signs and/orsymptoms of a disease or disorder being treated, e.g., reduces tumorsize or tumor burden. The actual amount administered can be determinedby an ordinarily skilled clinician based upon, for example, thesubject's age, weight, sex, general heath and tolerance to drugs,severity of disease, dosage form selected, route of administration, andother factors.

In specific aspects of the method, the subject has a cancer selectedfrom the group consisting of ovarian cancer, endometrial cancer,fallopian tube cancer, cervical cancer, breast cancer, lung cancer,mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophagealcancer, colon cancer, rectal cancer, and stomach cancer), pancreaticcancer, bladder cancer, kidney cancer, liver cancer, head and neckcancer, brain cancer, thyroid cancer, skin cancer, prostate cancer,testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML),and chronic myelogenous leukemia (CML).

The present disclosure also includes use of NDCs for treating a folatereceptor expressing tumor. For example, the use of NDC may compriseadministration to the subject in need thereof intravenously.

The present disclosure also relates to the use of NDCs in a subject withcancer selected from the group consisting of ovarian cancer, endometrialcancer, fallopian tube cancer, cervical cancer, breast cancer, lungcancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g.,esophageal cancer, colon cancer, rectal cancer, and stomach cancer),pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head andneck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer,testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML),and chronic myelogenous leukemia (CML).

The NDCs of the present disclosure may also be used in the manufactureof a medicament for treating a folate receptor expressing tumor, whereinthe NDC is administered to the subject in need thereof intravenously andwherein the subject has a cancer selected from the group consisting ofovarian cancer, endometrial cancer, fallopian tube cancer, cervicalcancer, breast cancer, lung cancer, mesothelioma, uterine cancer,gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectalcancer, and stomach cancer), pancreatic cancer, bladder cancer, kidneycancer, liver cancer, head and neck cancer, brain cancer, thyroidcancer, skin cancer, prostate cancer, testicular cancer, acute myeloidleukemia (AML, e.g., pediatric AML), and chronic myelogenous leukemia(CML).

The compositions and methods disclosed herein can include compositionsand methods that include administering a NDC as disclosed herein incombination with one or more additional anti-cancer agents. In suchcircumstances the NDC can be administered before, substantiallyconcurrently with, or after the additional agent or agents. Suitableadditional agents, include, for example chemotherapeutic agents such asmechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide,busulfan, N-nitroso-N-methylurea, carmustine, lomustine, semustine,fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide,thiotepa, mitomycin, diaziquone, cisplatin, carboplatin, oxaliplatin,procarbazine, hexamethylmelamine, methotrexate, pemetrexed, fluorouracil(e.g. 5-fluorouracil), capecitabine, cytarabine, gemcitabine,decitabine, azacitidine, fludarabine, nelarabine, cladribine,clofarabine, pentostatin, thioguanine, mercaptopurine, vincristine,vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel,irinotecan, topotecan, camptothecin, etoposide, mitoxantrone,teniposide,novobiocin, merbarone, doxorubicin, daunorubicin, epirubicin,idarubicin, pirarubicin, aclarubicin, mitomycin C, actinomycin,bleomycin, bisantrene, gemcitabine, cytarabine, and the like. Otheranti-cancer agents that can be used with a NDC in the compositions andmethods disclosed herein include, immune check point inhibitors (e.g.,anti-PD1, anti-PDL1, anti-CTLA4 antibodies), hormone receptorantagonists, other chemotherapeutic conjugates (e.g., in the form ofantibody-drug conjugates, nanoparticle drug conjugates, and the like),and the like.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe embodiments of the invention and the appended claims, the singularforms of “a”, “an” and “the” are intended to include the plural forms aswell, unless the context clearly indicates otherwise. Also, as usedherein, “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items.

The term “about,” when referring to a value means ±20%, or ±10. Further,the term “about” when used in connection with one or more numbers ornumerical ranges, should be understood to refer to all such numbers,including all numbers in a range and modifies that range by extendingthe boundaries above and below the numerical values set forth. Therecitation of numerical ranges by endpoints includes all numbers, e.g.,whole integers, including fractions thereof, subsumed within that range(for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, aswell as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) andany range within that range.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

It will be understood that in the detailed description and appendedclaims, the abbreviations and nomenclature employed are those which arestandard in amino acid and peptide chemistry.

Abbreviations

The abbreviations used in this disclosure, unless otherwise indicatedare as follows:

-   Fmoc: Fluorenylmethoxycarbonyl-   MeOH: Methanol-   Cit-OH: L-Citrulline-   DCM: Dichloromethane-   EEDQ: 2-Ethoxy-1-(ethoxycarbonyl)-1,2-dihydroquinoline-   THF: Tetrahydrofuran-   NMR: Nuclear Magnetic Resonance-   DMSO: Dimethyl sulfoxide-   LCMS: Liquid Chromatography-Mass Spectrometry-   TEA: Triethylamine-   HATU:    (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium    3-oxide hexafluorophosphate-   DMF: Dimethylformamide-   DIPEA: N,N-Diisopropylethylamine-   TMSCN: Trimethylsilyl cyanide-   RP HPLC: Reverse Phase High-Pressure Liquid Chromatography-   SFC: Supercritical fluid chromatography-   CAN: Acetonitrile-   NMP: N-Methyl pyrrolidone-   r.t: Room Temperature-   TEA: Triethylamine-   TFA: Trifluoroacetic acid-   MTBE: Methyl tert-butyl ether-   EtOAC: Ethyl acetate-   PyBOP:    (Benzotrizole-1-yl-oxytripyrrolidinenophosphoniumhexafluorophosphate)

Definitions

As used herein, the term “alkyl” refers to monovalent aliphatichydrocarbon group that may comprise 1 to 18 carbon atoms, such as 1 toabout 12 carbon atoms, or 1 to about 6 carbon atoms (“C₁₋₁₈ alkyl”). Analkyl group can be straight chain, branched chain, monocyclic moiety orpolycyclic moiety or combinations thereof. Examples of alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl,tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. Each instance of an alkyl group maybe independently optionally substituted, i.e., unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents e.g., for instance from 1 to 5 substituents, 1 to 3substituents, or 1 substituent.

As used herein, the term “alkenyl” refers to a monovalent straight-chainor branched hydrocarbon group having from 2 to 18 carbon atoms, one ormore carbon-carbon double bonds, and no triple bonds (“C₂₋₁₈ alkenyl”).An alkenyl group may have 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. The one ormore carbon-carbon double bonds can be internal (such as in 2-butenyl)or terminal (such as in 1-butenyl). Examples of alkenyl groups includeethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, butadienyl,pentenyl, pentadienyl, hexenyl, heptenyl, octenyl, octatrienyl, and thelike. Each instance of an alkenyl group may be independently optionallysubstituted, i.e., unsubstituted (an “unsubstituted alkenyl”) orsubstituted (a “substituted alkenyl”) with one or more substituentse.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1substituent.

As used herein, the term “alkynyl” refers to a monovalent straight-chainor branched hydrocarbon group having from 2 to 18 carbon atoms, one ormore carbon-carbon triple bonds (“C2-18 alkynyl”). The alkynyl group mayhave 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to4 carbon atoms, or 2 to 3 carbon atoms. The one or more carbon-carbontriple bonds can be internal (such as in 2-butynyl) or terminal (such asin 1-butynyl). Examples of alkynyl groups include ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl, and the like. Each instance of analkynyl group may be independently optionally substituted, i.e.,unsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents, e.g., for instancefrom 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

As used herein, the term “heteroalkyl” refers to a non-cyclic stablestraight or branched chain, or combinations thereof, including at leastone carbon atom and at least one heteroatom selected from the groupconsisting of O, N, P, Si, and S, and wherein the nitrogen and sulfuratoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, P, S, and Si may beplaced at any position of the heteroalkyl group.

The terms “alkylene,” “alkenylene,” “alkynylene,” or “heteroalkylene,”alone or as part of another substituent, mean, unless otherwise stated,a divalent radical derived from an alkyl, alkenyl, alkynyl, orheteroalkyl, respectively. The term “alkenylene,” by itself or as partof another substituent, means, unless otherwise stated, a divalentradical derived from an alkene. An alkylene, alkenylene, alkynylene, orheteroalkylene group may be described as, e.g., a C₁₋₆-memberedalkylene, C₁₋₆-membered alkenylene, C₁₋₆-membered alkynylene, orC₁₋₆-membered heteroalkylene, wherein the term “membered” refers to thenon-hydrogen atoms within the moiety. In the case of heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(0)2R′— may represent both —C(O)₂R′— and —R′C(O)₂—. Eachinstance of an alkylene, alkenylene, alkynylene, or heteroalkylene groupmay be independently optionally substituted, i.e., unsubstituted (an“unsubstituted alkylene”) or substituted (a “substitutedheteroalkylene”) with one or more substituents.

As used herein, the terms “substituted alkyl,” “substituted alkenyl,”“substituted alkynyl,” “substituted heteroalkyl,” “substitutedheteroalkenyl,” “substituted heteroalkynyl,” “substituted cycloalkyl,”“substituted heterocyclyl,” “substituted aryl,” and “substitutedheteroaryl” refer to alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, andheteroaryl moieties, respectively, having substituents replacing one ormore hydrogen atoms on one or more carbons or heteroatoms of the moiety.Such substituents can include, for example, alkyl, alkenyl, alkynyl,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Cycloalkyls can be further substituted, e.g., with the substituentsdescribed above.

As used herein, the term “alkoxy” refers to a group of formula —O—alkyl. The term “alkoxy” or “alkoxyl” includes substituted andunsubstituted alkyl, alkenyl and alkynyl groups covalently linked to anoxygen atom. Examples of alkoxy groups or alkoxyl radicals include, butare not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy andpentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

As used herein, the term “aryl,” refers to stable aromatic ring system,that may be monocyclic or polycyclic, of which all the ring atoms arecarbon, and which may be substituted or unsubstituted. The aromatic ringsystem may have, for example, 3-7 ring atoms. Examples include phenyl,benzyl, naphthyl, anthracyl, and the like. Each instance of an arylgroup may be independently optionally substituted, i.e., unsubstituted(an “unsubstituted aryl”) or substituted (a “substituted aryl”) with oneor more substituents.

As used herein, the term “heteroaryl” refers to an aryl group thatincludes one or more ring heteroatoms. For example, a heteroaryl caninclude a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, or9-membered bicyclic aromatic heterocyclic ring which consists of carbonatoms and one or more heteroatoms, independently selected from the groupconsisting of nitrogen, oxygen and sulfur. The nitrogen atom may besubstituted or unsubstituted (e.g., N or NR₄ wherein R₄ is H or othersubstituents, as defined). Examples of heteroaryl groups includepyrrole, furan, indole, thiophene, thiazole, isothiazole, imidazole,triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine,pyridazine, pyrimidine, and the like.

As used herein, the terms “cycloalkylene,” “heterocyclylene,” “arylene,”and “heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from a cycloalkyl, heterocyclyl, aryl, andheteroaryl, respectively. Each instance of a cycloalkylene,heterocyclylene, arylene, or heteroarylene may be independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted arylene”)or substituted (a “substituted heteroarylene”) with one or moresubstituents.

As used herein, the term “cycloalkyl”, is intended to includenon-aromatic cyclic hydrocarbon rings, such as hydrocarbon rings havingfrom three to eight carbon atoms in their ring structure. Cycloalkyl caninclude cyclobutyl, cyclopropyl, cyclopentyl, cyclohexyl and the like.The cycloalkyl group can be either monocyclic (“monocyclic cycloalkyl”)or contain a fused, bridged or spiro ring system such as a bicyclicsystem (“bicyclic cycloalkyl”) and can be saturated or can be partiallyunsaturated. “Cycloalkyl” also includes ring systems wherein thecycloalkyl ring, as defined above, is fused with one or more aryl groupswherein the point of attachment is on the cycloalkyl ring, and in suchinstances, the number of carbons continue to designate the number ofcarbons in the cycloalkyl ring system. Each instance of a cycloalkylgroup may be independently optionally substituted, i.e., unsubstituted(an “unsubstituted cycloalkyl”) or substituted (a “substitutedcycloalkyl”) with one or more substituents.

As used herein, the term “heterocyclyl” refers to a monovalent cyclicmolecular structure comprising atoms of at least two different elementsin the ring or rings (i.e., a radical of a heterocyclic ring).Additional reference is made to: Oxford Dictionary of Biochemistry andMolecular Biology, Oxford University Press, Oxford, 1997 as evidencethat heterocyclic ring is a term well-established in field of organicchemistry.

As used herein, the term “dipeptide” refers to a peptide that iscomposed of two amino-acid residues, that may be denoted herein as-A₁-A₂-. For example, dipeptides employed in the synthesis ofprotease-cleavable linker-payload conjugates of the present disclosuremay be selected from the group consisting of Val-Cit, Phe-Lys, Trp-Lys,Asp-Lys, Val-Lys, and Val-Ala.

As used herein, the term “functionalized polyethylene glycol” refers tothe polyethylene glycol comprising a functional group. For example, afunctionalized polyethylene glycol may be polyethylene glycolfunctionalized with a terminal group selected from the group consistingof azide,

wherein R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl. In preferred aspects,R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence ishydrogen. In preferred aspects, R^(1′), R^(2′), R^(3′), R^(4′) andR^(5′) in each occurrence is methyl.

In some aspects of the present disclosure, the term “functionalizedpolyethylene glycol” refers to, but is not limited to the followingstructures.

As used herein, Ti may refer to a functionalized polyethylene glycol ora C₅-C₆ alkyl chain that has a terminal group selected from the groupconsisting of azide,

wherein R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl. In preferred aspects ofT¹, R^(1′), R^(2′), R^(3′), R⁴′ and R^(5′) in each occurrence ishydrogen. In preferred aspects of T¹, R^(1′), R^(2′), R^(3′), R^(4′) andR⁵′ in each occurrence is methyl. In preferred aspects, T₁ is afunctionalized polyethylene glycol that has an azide terminal group. Inpreferred aspects, T₁ is a C₅-C₆ alkyl chain that has an azide terminalgroup. The repeat unit (—O—CH₂—CH₂—) of polyethylene glycol (PEG) canrange from 5-20 units, preferably 5-15 units and more preferably 6-12.

As used herein, T₁ may refer to a C₅-C₆ alkyl chain that has a terminalgroup selected from the group consisting of azide,

wherein R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in each occurrence isindependently hydrogen, substituted or unsubstituted C₁₋₆ alkyl orsubstituted or unsubstituted C₁₋₆ cycloalkyl. In preferred aspects,R^(1′), R^(2′), R^(3′), R⁴′ and R^(5′) in each occurrence is hydrogen.In preferred aspects, R^(1′), R^(2′), R^(3′), R^(4′) and R^(5′) in eachoccurrence is methyl.

Monofunctionalized azide-terminated PEG and monofunctionalizedazide-terminated C₅-C₆ alkyl chain can be made from PEG using knownprocedures and suitable reagents, such as those disclosed in the Schemesprovided herein.

As used herein, the term “halo” or “halogen” refers to F, Cl, Br, or I.

An aryl or heteroaryl group described herein can be substituted at oneor more ring positions with such substituents as described above, forexample, alkyl, alkenyl, akynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

As used herein, the term “hydroxy” refers to a group of formula —OH.

As used herein, the term “hydroxyl” refers to a hydroxyl radical (.0H).

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. In general, the term “substituted” means that at leastone hydrogen present on a group (e.g., a carbon or nitrogen atom) isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound. The term “substituted” caninclude substitution with all permissible substituents of organiccompounds, such as any of the substituents described herein that resultin the formation of a stable compound. For purposes of the presentdisclosure, heteroatoms such as nitrogen may have hydrogen substituentsand/or any suitable substituent as described herein which satisfy thevalencies of the heteroatoms and results in the formation of a stablemoiety.

As used herein, the term “tetrapeptide” refers to a peptide that iscomposed of four amino-acid residues, that may be denoted herein as-A₁-A₂-A₃-A₄-. Tetrapeptides employed in the synthesis ofprotease-cleavable linker-payload conjugates of the present disclosureis selected from the group consisting of Val-Phe-Gly-Sar,Val-Cit-Gly-Sar, Val-Lys-Gly-Sar, Val-Ala-Gly-Sar, Val-Phe-Gly-Pro,Val-Cit-Gly-Pro, Val-Lys-Gly-Pro, Val-Ala-Gly-Pro, Val-Cit-Gly-anynatural or unnatural N-alkyl substituted alpha amino acid,Val-Lys-Gly-any natural or unnatural N-alkyl substituted alpha aminoacid, Val-Phe-Gly-any natural or unnatural N-alkyl substituted alphaamino acid, Val-Ala-Gly-any natural or unnatural N-alkyl substitutedalpha amino acid, Phe-Lys-Gly-any natural or unnatural N-alkylsubstituted alpha amino acid, and Trp-Lys-Gly-any natural or unnaturalN-alkyl substituted alpha amino acid.

Furthermore, it will be appreciated by one of ordinary skill in the artthat the synthetic methods, as described herein, utilize a variety ofprotecting groups. As used herein, the term “protecting group” refers toa particular functional moiety, e.g., O, S, or N, that is temporarilyblocked so that a reaction can be carried out selectively at anotherreactive site in a multifunctional compound. Protecting groups may beintroduced and removed at appropriate stages during the synthesis of acompound using methods that are known to one of ordinary skill in theart. The protecting groups are applied according to standard methods oforganic synthesis as described in the literature (Theodora W. Greene andPeter G. M. Wuts (2007) Protecting Groups in Organic Synthesis, 4^(th)edition, John Wiley and Sons, incorporated by reference with respect toprotecting groups).

Exemplary protecting groups include, but are not limited to, oxygen,sulfur, nitrogen and carbon protecting groups. For example, oxygenprotecting groups include, but are not limited to, methyl ethers,substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM(methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM(pimethoxybenzyloxymethyl ether), optionally substituted ethyl ethers,optionally substituted benzyl ethers, silyl ethers (e.g., TMS(trimethylsilyl ether), TES (triethylsilylether), TIPS(triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzylsilyl ether, TBDPS (t-butyldiphenyl silyl ether), esters (e.g., formate,acetate, benzoate (Bz), trifluoroacetate, dichloroacetate) carbonates,cyclic acetals and ketals. In addition, nitrogen protecting groupsinclude, but are not limited to, carbamates (including methyl, ethyl andsubstituted ethyl carbamates (e.g., Troc), amides, cyclic imidederivatives, N-Alkyl and N-Aryl amines, imine derivatives, and enaminederivatives, etc. Amino protecting groups include, but are not limitedto fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc),carboxybenzyl (Cbz), acetamide, trifluoroacetamide, etc. Certain otherexemplary protecting groups are detailed herein, however, it will beappreciated that the present disclosure is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups may be utilized according to methods known to oneskilled in the art.

Throughout this disclosure, a nanoparticle-drug-conjugate (NDC) maysometimes be referred to as a CDC (C'Dot-drug-conjugate), e.g., aFA-CDC.

The following examples are provided to further illustrate theembodiments of the present invention but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. These examples areoffered to illustrate the nanoparticle drug conjugates, methods of use,and methods of making, and are not to be construed in any way aslimiting their scope.

The compounds provided herein can be prepared from readily availablestarting materials using modifications to the specific synthesisprotocols set forth below that would be well known to those of skill inthe art. It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvents used, butsuch conditions can be determined by those skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice ofsuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in Greene et al. Protecting Groups in OrganicSynthesis, Second Edition, Wiley, New York, 1991, and references citedtherein.

General Methods

Methods useful for making the compounds discussed herein are set forthin the following Examples and are generalized here. One of skill in theart will recognize that these Examples can be adapted to prepare thelinker-payload conjugates, linkers and payloads and theirpharmaceutically accepted salts thereof according to the presentdisclosure. In the reactions described, reactive functional groups, suchas hydroxy, amino, imino, thio or carboxy groups, may be protectedwherever desired, e.g., to avoid unwanted reactions. Conventionalprotecting groups may also be used in accordance with standard practiceand techniques of synthesis. The materials needed to synthesize thenovel linkers bearing payloads such as exatecan were obtainedcommercially, and their corresponding analogs are prepared as disclosedin the following examples.

Reagents were purchased from commercial suppliers(Combi-Blocks/SIGMA-ALDRICH) and used without further purification. Allnon-aqueous reactions were run in flame-dried glassware under a positivepressure of argon. Anhydrous solvents were purchased from commercialsuppliers (RANKEM). All the amino acids such as Cit, Val, Phe, Lys, Trp,Asp are naturally occurring amino acids with S-configuration. In severalexamples, tetrapeptide and unnatural amino acids can also be used. Flashchromatography was performed on 230-400 mesh silica gel with theindicated solvent systems. Proton Nuclear magnetic resonance spectrawere recorded on Bruker Spectrometer at 400MHZ using DMSO as solvent.Peak positions are given in parts per million downfield fromtetramethylsilane as the internal standard. J values are expressed inhertz. Mass analyses were performed on (Agilent/Shimadzu) spectrometerusing electrospray (ES) technique. HPLC analyses were performed on(Agilent/Waters), PDA-UV detector equipped with a Gemini C-18 (1000×4.6mm; 5u) and all compounds tested were determined to be >95% pure usingthis method. As can be seen in many protease-cleavable linker-payloadconjugates, two peaks were isolated at the end of the reaction. ThePeak-A (or Peak-1) is the desired compound with the stereochemistry asshown.

Compounds prepared according to the procedures described herein may beisolated by preparative HPLC methods. Representative HPLC conditions andmethods are provided below:

Agilent UPLC-MS; Column: Column-YMC Triart C18 (2.1×33 mm, 3u)

Gradient Conditions: Flow rate: 1.0 ml/min; column temperature: 50° C.;Solvent A: 0.01% HCOOH in water and Solvent B: 0.01% HCOOH in CH3CN;Mobile phase: 95% [0.01% HCOOH in water] and 5% [0.01% HCOOH in CH₃CN]held for 0.50 min, then to 1% [0.01% HCOOH in water] and 99% [0.01%HCOOH in CH₃CN] in 3.00 min, held this conditions up to 4.00 min andfinally back to initial condition in 4.10 min and held for 4.50 min(Table 1).

TABLE 1 HPLC Gradient Conditions. TIME MODULE % A % B 0.00 Pumps 95 50.50 Pumps 95 5 3.00 Pumps 1 99 4.00 Pumps 1 99 4.10 Pumps 95 5 4.50Pumps 95 5

Example 1 Synthesis of Exatecan-Linker Conjugate Precursors

Exatecan-linker conjugate precursors suitable for preparing an NDC ofthe present disclosure can be synthesized according to the followingprotocols. As the exatecan-linker conjugate precursors comprise aterminal azide group, they are suitable for attaching to a nanoparticlefunctionalized with alkyne moieties (e.g., DBCO), using click chemistry.Synthesis of(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(ptolyl)methyl)amino)hexanamide(161)

Synthesis of 4-(((tent-butyldiphenylsilyl)oxy)methyl)aniline (159):Imidazole (5.54 g, 81.22 mmol) was added to a solution of(4-aminophenyl)methanol (75) (5.0 g, 40.61 mmol) in DMF (25 mL) at 0°C., followed by tert-butyl(chloro)diphenylsilane (13.39 g, 48.73 mmol),and the reaction mixture was stirred at room temperature for 16 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was quenched with water (20 mL)and extracted with EtOAc (2×200 mL). The combined organic layers weredried over anhydrous Na₂SO₄, concentrated under reduced pressure, andpurified by column chromatography using silica gel (230-400 mesh)eluting with 10% EtOAc in petroleum ether to afford4-(((tert-butyldiphenylsilyl)oxy)methyl)aniline (159; 6.6 g) as a gum.LCMS: m/z 362.31 [(M+H)⁺]; R_(t): 2.58 min; 93.68% purity.

Synthesis of(9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(160): Diisopropylethylamine (4.18 mL, 24 mmol), HATU (6.08 g, 16 mmol)and 4-(((tert-butyldiphenylsilyl)oxy)methyl)aniline (159) (2.89 g, 8mmol) were added to a solution ofN2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(diphenyl(p-tolyl)methyl)-L-lysine(149) (5.0 g, 8 mmol) in DMF (50 mL)at 0° C., and the reaction mixturewas stirred at room temperature for 16 h. The progress of the reactionwas monitored by TLC. After completion of starting material, thereaction mixture was quenched with ice water. The precipitated solid wasfiltered and dried under vacuum to afford (9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(160; 5.5 g) as a solid. LCMS: m/z 990.37 [(M+H)⁺]; R_(t): 2.84 min;96.79% purity.

Synthesis of(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(ptolyl)methyl)amino)hexanamide (161): Piperidine (16.5 mL) was added to asolution of (9H-fluoren-9-yl)methyl(S)-(1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)carbamate(160) (5.5 g, 5.68 mmol) in DMF (38.5 mL) at room temperature, and thereaction mixture was stirred at room temperature for 3 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was concentrated under reduced pressure,and purified by column chromatography using silica gel (230-400 mesh)eluting with 100% EtOAc to afford(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(160; 3.5 g) as a gum. LCMS: m/z 744.24 [(M−H)⁻]; R_(t): 2.20 min;90.16% purity.

Synthesis of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(4-nitrophenyl) carbonate (191).

Synthesis of(9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(187): Diisopropylethylamine (1.54 mL, 8.83 mmol), HATU (2.24 g, 5.89mmol) and(S)-2-amino-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyhp-tolyl)methyl)amino)hexanamide(161) (2.19 g, 2.94 mmol) were added to a solution of(((N-(9-Fluorenylmethoxycarbonyl)-L-valine (1 g, 2.94 mmol), in DMF (20mL)at 0° C., and the reaction mixture was stirred at room temperaturefor 3 h. The progress of the reaction was monitored by TLC. Aftercompletion of starting material, the reaction mixture was quenched withice water. The precipitated solid was filtered and dried under vacuum toafford (((9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(187; 2.5 g) as a solid. LCMS: MH⁺ 1067, retention time 2.42 min.

Synthesis of(S)-2-((S)-2-Amino-3-methylbutanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyl(p-tolyl)methyl)amino)hexanamide(188): A 30% solution of piperidine in DMF (4.5 mL) was added to asolution of (((9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(187) (1.5 g, 1.40 mmol) in DMF (6 mL) at room temperature, and thereaction mixture was stirred at room temperature for 2 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was concentrated under reduced andpurified by flash chromatography eluting with 100% EtOAc, to afford(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyhp-tolyl)methyl)amino)hexanamide(188; 1.1 g) as a solid. ¹H NMR (400 MHz, DMSO-d₆): δ 10.07 (s, 1H),7.64-7.63 (d, 4H), 7.56-7.54 (d, 2H), 7.46-7.35 (m, 9H), 7.27-7.24 (m,8H), 7.185-7.11 (m, 2H), 7.05-7.03 (d, 2H), 4.71 (s, 2H), 4.44 (d, 1H),3.25-3.16 (d, 1H), 3.01-3.00 (m, 1H), 2.21 (s, 3H), 1.98-1.93 (m, 2H),1.68-1.38 (m, 4H), 1.15 (s, 10H), LCMS: MH⁺ 845, retention time 3.63min.

Synthesis of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(189): Diisopropylethylamine (0.49 mL, 2.83 mmol), HATU (719.47 mg, 1.89mmol) and(S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)-6-((diphenyhp-tolyl)methyl)amino)hexanamide(188) (800 mg, 0.94 mmol) were added to a solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86) (484mg, 0.94 mmol) in DMF (8 mL)at 0° C., and the reaction mixture wasstirred at room temperature for 6 h. The progress of the reaction wasmonitored by TLC. After completion of starting material, the reactionmixture was quenched with water (15 mL) and extracted with EtOAc (2×30mL). The combined organic layers were dried over anhydrous Na₂SO₄,concentrated under reduced, and purified by flash chromatography elutingwith 3% MeOH in DCM to provide1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyOmethyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(189; 0.60 g) as a gum. ¹H NMR (400 MHz, DMSO-d₆): δ 9.91 (s, 1H),8.02-8.00 (d, 2H), 7.95 (s, 2H), 7.87-7.85 (d, 1H), 7.64-7.63 (d, 4H),7.57-7.55 (d, 2H), 7.46-7.32 (m, 11H), 7.26-7.24 (m, 8H), 7.15-7.11 (t,2H), 7.05-7.03 (d, 2H), 4.71 (s, 2H), 4.35-4.33 (m, 1H), 4.19 (s, 1H),3.59-3.36 (m, 38H), 2.68-2.38 (m, 6H), 2.22 (s, 3H), 1.98-1.92 (m, 2H),1.47-1.17 (m, 4H), 1.02 (s,9H), 0.85-0.80 (m, 6H). LCMS: MH⁺ 1338,retention time 2.92 min.

Synthesis of1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-l-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(190): NH₄F (166 mg, 4.48 mmol) was added to a solution of1-azido-N-((S)-1-(((S)-1-((4-(((tert-butyldiphenylsilyl)oxy)methyl)phenyl)amino)-6-((diphenyl(p-tolyl)methyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(189) (600 mg, 0.44 mmol) in methanol (10 mL) at room temperature, andthe reaction mixture was stirred at room temperature for 6 h. Theprogress of the reaction was monitored by TLC. After completion ofstarting material, the reaction mixture was concentrated under reducedpressure, and the residue obtained was diluted with water (15 mL) andextracted with EtOAc (2×20 mL). The combined organic layers were driedover anhydrous Na₂SO₄, concentrated under reduced pressure, and purifiedby flash chromatography eluting with 5% MeOH in DCM to afford1-azido-N-((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(190; 0.40 g) as a gum. ¹H NMR (400 MHz, DMSO-d₆): δ 9.81 (s, 1H),7.96-7.94 (d, 1H), 7.84-7.81 (d, 1H), 7.53-7.51 (d, 2H), 7.37-7.35 (d,4H), 7.26-7.12 (m, 9H), 7.096-7.04 (d, 2H), 5.06-5.04 (t, 1H), 4.43-4.41(d, 2H), 4.35 (m, 1H), 4.18-4.16 (t, 1H), 3.60-3.46 (m, 33H), 3.39-3.36(t, 2H), 2.50-2.23 (m, 2H), 2.23 (s, 3H), 2.23-1.93 (m,2H), 1.48-1.23(m, 6H), 0.85-0.80 (m, 6H). LCMS: MH⁺ 1100, retention time 3.72 min.

Synthesis of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(4-nitrophenyl) carbonate (191): Pyridine (0.14 mL, 1.80 mmol) and4-nitrophenyl chloroformate (14) (145 mg, 0.72 mmol) were added to asolution of1-azido-N—((S)-1-(((S)-6-((diphenyl(p-tolyl)methyl)amino)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxohexan-2-y0amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(190) (400 mg, 0.36 mmol) in DCM (10 mL)at 0° C., and the reactionmixture was stirred at room temperature for 6 h. The progress of thereaction was monitored by TLC. After completion of starting material,the reaction mixture was concentrated under reduced pressure, andpurified by flash chromatography eluting with 3% MeOH in DCM to afford4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(4-nitrophenyl) carbonate (191; 0.34 g) as a gum. LCMS: MH⁺ 1265,retention time 1.33 min.

Synthesis of4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-l-yl)carbamate(202).

Synthesis of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(201): Triethylamine (0.09 mL, 0.62 mmol) and(1R,9R)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dionemethanesulfonate (exatecan mesylate; 16; 131 mg, 0.25 mmol) were addedto a solution4-((32S,35S)-1-azido-35-(4-((diphenyhp-tolyOmethyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl(4-nitrophenyl) carbonate (191; 311 mg, 0.25 mmol) in NMP (2.5 mL) at 0°C., and the mixture was stirred at room temperature for 8 h. Theprogress of the reaction was monitored by LCMS. After completion ofstarting material, the reaction mixture was quenched with water (15 mL)and extracted with 10% methanol in chloroform (2×20 mL). The combinedorganic layers were dried over anhydrous sodium sulfate (Na₂SO₄) andconcentrated under reduced pressure. Diethyl ether was added to thecrude material, and the resulting precipitate was filtered and purifiedusing column chromatography (Combi-Flash) eluting with 5% MeOH in DCM toprovide4-((32S,35S)-1-azido-35-(4-((diphenyhp-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(201) as a solid (0.3 g). LCMS: MH⁺ 1561, retention time 2.18 min.

Synthesis of4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(202): A 1% solution of trifluoroacetic acid (TFA) in DCM was added to asolution of4-((32S,35S)-1-azido-35-(4-((diphenyl(p-tolyl)methyl)amino)butyl)-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(201; 300 mg, 0.19 mmol) in DCM (5 mL) at 0° C., and the reactionmixture was stirred at room temperature for 1 h. The progress of thereaction was monitored by LCMS. After completion of starting material,the reaction mixture was concentrated under reduced pressure, and theresidue was triturated with diethyl ether and purified by RP-prep-HPLCto provide4-((32S,35S)-35-(4-aminobutyl)-1-azido-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazahexatriacontan-36-amido)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate(202) (70mg) as a solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.96 (s, 1H),8.12-8.10 (q, 2H), 7.89-7.87 (d, 1H), 7.76-7.61 (d, 1H), 7.59-7.31 (m,7H), 6.51 (s, 1H), 5.44 (s, 2H), 5.29 (s, 3H), 5.09 (s, 2H), 4.37-4.20(m, 1H), 4.18-4.16 (t, 1H), 3.49-3.44 (m, 4H), 3.12-2.55 (m, 39H),2.40-1.34 (m, 15H), 0.89-0.82 (m, 9H), LCMS: MEI⁺ 1305, retention time5.33 and 5.47 min.

Example 2 Synthesis of Folic Acid Conjugate Precursors

Folic acid conjugate precursors suitable for preparing a folate receptortargeting NDC disclosed herein can be prepared according to one of thefollowing synthetic protocols. As the folic acid conjugate precursorscomprise a terminal azide group, they are suitable for attaching to ananoparticle functionalized with alkyne moieties (e.g., DBCO), usingclick chemistry.

Synthesis of(S)-16-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-azido-13-oxo-3,6,9-trioxa-12-azaheptadecan-17-oicacid (606)

Preparation of compound 600: Compound 599 (160 g, 512 mmol) wasdissolved in TFAA (800 mL) at 25° C. and stirred under a nitrogenatmosphere in the dark for 5 hrs. The solvent was then removed at 50° C.in vacuo to give the crude product. The crude product was trituratedwith MTBE (750 mL) for 60 min and then filtered to afford compound 600(203 g, crude) as a solid, which was used in next step without furtherpurification. LC-MS: ¹H NMR: (400 MHz, CDCl₃) δ 12.74 (br s, 1H), 8.88(s, 1H), 7.97-8.05 (m, 2H), 7.66-7.74 (m, 2H), 5.26 (s, 1H).

Preparation of Compound 602: TBTU (238 g, 740 mmol) and DIPEA (95.7 g,740 mmol) were added to a solution of compound 601 (225 g, 529 mmol) inDMF (2.25 L). After 30 min stirring at 20° C.,2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (Reagent A; 121 g,555 mmol) was added and the mixture was stirred at 50° C. for 12 hrs.Two reaction mixtures were combined and worked up, and the residue wasdiluted with H₂O (3 L) and extracted with ethyl acetate (1500 mL×3). Thecombined organic layers were washed with brine (800 mL×3), dried overNa₂SO₄, filtered and concentrated under reduced pressure, and purifiedby column chromatography (SiO₂, Petroleum ether/Ethyl acetate=100/1 to1/1) to afford compound 602 (590 g) as an oil. ¹H NMR: (400 MHz, CDCl₃)δ 7.76-7.78 (m, 2H), 7.63-7.60 (m, 2H), 7.41-7.27 (m, 4H), 6.43 (s, 1H),5.70 (s, 1H), 4.42-4.38 (m, 2H), 4.24-4.23 (m, 2H), 3.63-3.36 (m, 16H),2.28-2.18 (m, 3H), 1.98-1.96 (m, 1H), 1.48 (s, 9H).

Preparation of Compound 603: N-ethylethanamine (1.27 kg, 17.4 mol) wasadded to a solution of compound 602 (435 g, 695 mmol) in DCM (4.35 L)and the mixture was stirred at 25° C. 3 hrs. The solvent was thenremoved at room temperature in vacuo, and the residue was purified byflash column chromatography (DCM/MeOH=100/1 to 1/1) to afford compound603 (245 g) as an oil. ¹H NMR: (400 MHz, CDCl₃) δ 6.55 (s, 1H),3.67-3.30 (m, 17H), 2.34-2.30 (m, 2H), 2.10-2.06 (m, 1H), 1.87 (s, 2H),1.77-1.73 (m, 1H), 1.44 (s, 9H).

Preparation of compound 604: TBTU (119 g, 372 mmol) and DIEA (160 g,1.24 mol) were added to a solution of compound 600 (101 g, 248 mmol) inDMF (900 mL) and the mixture was stirred for 30 minutes. Then compound603 (100 g, 248 mmol) in DMF (100 mL) was added. The mixture was stirredat 25° C. for 12 hrs. Two reaction mixtures were combined andconcentrated and the residue was diluted with H₂O (2.5 L) and extractedwith ethyl acetate (1 L×5). The combined organic layers were washed withbrine (600 mL×3), dried over Na₂SO₄, filtered and concentrated underreduced pressure to afford compound 4 (420 g, crude) as a solid, whichwas used in next step without further purification.

Preparation of compound 605: K₂CO₃ (585 g, 4.23 mol) was added to asolution of compound 604 (420 g, 529 mmol) in THF (4.2 mL) and H₂O (500mL) and the mixture was stirred at 60° C. for 0.5 hr.. The reactionmixture was concentrated under reduced pressure to remove THF and theresidue was diluted with H₂O (500 mL) and adjusted the pH to 3 with HCl(M=1), filtered and concentrated under reduced pressure to affordcompound 605 (260 g, crude) as a solid, which was used directly withoutpurification.

Preparation of compound 606: Trifluoroacetic acid (2.12 kg, 18.6 mol)was added in one portion to a mixture of compound 605 (260 g, 373 mmol)in CH₂Cl₂ (2.6 L) at 20° C. under nitrogen, and the mixture was stirredat 20° C. for 5 hrs. The reaction mixture was concentrated under reducedpressure and purified by HPLC (column: Agela DuraShell C18 250*80 mm*10um; mobile phase: [water (10 mM NH4HCO3)-MeOH]; B%: 5%-40%,20 min) togive afford compound 606 (52.5 g, 81.82 mmol, 21.96% yield) as a solid.(M+H) 642.80; IR: 2107 (N₃ Bond).

Synthesis of(S)-38-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oicacid (472)

Synthesis of tent-butyl(1-azido-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatritriacontan-33-yl)carbamate(465): Triethylamine (0.36 mL, 2.64 mmol), EDC (218 mg, 1.14 mmol), HOBT(154 mg, 1.14 mmol) and tert-butyl (2-aminoethyl)carbamate (464) (124mg, 0.881 mmol) were added to a solution of1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-oic acid (86; 450mg, 0.881 mmol), in DCM (20 mL) at 0° C., and the reaction mixture wasstirred at room temperature for 16 h. The progress of the reaction wasmonitored by TLC. After consumption of starting material, the reactionmixture was extracted with DCM and water, and the organic layer wasdried over Na₂SO₄, and evaporated under vacuum. The residue was purifiedby flash chromatography and dried under vacuum to provide tert-butyl(1-azido-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatritriacontan-33-yl)carbamate(465; 0.45 g) as a liquid. ¹H NMR (400 MHz, DMSO-d₆): 7.83 (t, 1H), 6.75(t, 1H), 3.61-3.31(m, 38H), 3.02-2.97 (t, 4H), 2.28 (t, 2H), 1.37(s,9H).

Synthesis ofN-(2-Aminoethyl)-1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(466): A solution of tert-butyl(1-azido-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatritriacontan-33-yl)carbamate(465) (350 mg, 0.462 mmol) in DCM was cooled to 0° C. and TFA was addedto it by dropwise, and the reaction mixture was then stirred at RT for16 h. The progress of the reaction was monitored by TLC. Afterconsumption of starting material, the reaction mixture was concentratedunder reduced pressure and azeotroped with DCM (3 times) to providecrude (466), which was purified by flash chromatography eluting with 5%MeOH in DCM to provideN-(2-aminoethyl)-1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(466; 0.25 g) as a liquid. ¹H NMR (400 MHz, DMSO-d₆): 8.02 (t, 1H), 7.73(t, 2H), 3.71-3.26 (m, 40H), 2.86(t, 2H), 2.35(t, 2H).

Synthesis of tent-butyl(S)-38-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(467): Diisopropylethylamine (0.174 mL, 1.0 mmol), PyBOP (416 mg, 0.8mmol) andN-(2-aminoethyl)-1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide(466) (331mg, 0.6 mmol) were added to a solution of(S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoicacid (170 mg, 0.4 mmol), in DMF (5 mL) at 0° C., and the reactionmixture was stirred at rt for 16 h. The progress of the reaction wasmonitored by TLC. After consumption of starting material, the reactionmixture was evaporated under vacuum at low temperature, and purified byflash chromatography eluting with 5% MeOH in DCM to afford tert-butyl(S)-38-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(467; 0.35 g) as a liquid. MH⁺ 962, retention time 1.81 min.

Synthesis of tent-butyl(S)-38-amino-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(468): A 30% solution of piperidine in DMF (1 ml) was added to asolution of tert-butyl(S)-38-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(467; 350mg, 3.65 mmol) in DMF (5 mL) at room temperature, and thereaction mixture was stirred at room temperature for 3 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was concentrated under reduced pressureto afford tert-butyl(S)-38-amino-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(468; 250 mg), which was used in the next step without furtherpurification. MH⁺ 739, retention time 1.50 min.

Synthesis of tert-butyl(S)-38-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2, 2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(470): Diisopropylethylamine (0.107 mL, 0.613 mmol), PyBOP (254 mg, 0.49mmol) and tert-butyl(S)-38-amino-l-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(468) (271 mg, 0.368 mmol) were added to a solution of4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzoicacid (469;100mg, 0.245 mmol) in DMF (5 mL) at 0° C., and the reactionmixture was stirred at room temperature for 16 h. The progress of thereaction was monitored by TLC. After completion of starting material,the reaction mixture was concentrated under vacuum at low temperature,then purified by flash chromatography eluting with 10% MeOH in DCM toafford tert-butyl(S)-38-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(470; 180 mg) as a solid. MH⁺ 1129, retention time 2.61 min.

Synthesis of(S)-38-(4-(N-((2-Amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oicacid (471): Trifluoroacetic acid (0.123 mL, 1.59 mmol) was added to asolution of tert-butyl(S)-38-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oate(470) (180 mg, 0.16 mmol) in DCM was added at room temperature, and thereaction mixture was stirred at room temperature for 16 h. The progressof the reaction was monitored by TLC. After completion of startingmaterial, the reaction mixture was concentrated under reduced pressureand azeotroped with DCM (3 times) to afford crude product (471; 100 mg),that was used in the next step without further purification. MH⁺ 1073,retention time 2.34 min.

Synthesis of(S)-38-(4-(((2-Amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oicacid (472): Aqueous NH₃ (dissolved in DMF) (0.01 mL, 0.71 mmol) wasadded to a solution of(S)-38-(4-(N-((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)-2,2,2-trifluoroacetamido)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oicacid (471; 80 mg, 0.071 mmol) in DMF (3mL) at 0° C., and the reactionmixture was stirred at room temperature for 6 h. After completion ofstarting material, the reaction mixture was concentrated under reducedpressure, and the residue was purified by RP-prep-HPLC to afford(S)-38-(4-(((2-Amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-azido-30,35-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31,34-diazanonatriacontan-39-oicacid (472; 15 mg) as a solid. ¹H NMR (400 MHz, DMSO-d₆): 8.62 (S, 1H),8.01(d, 1H), 7.98(t,1H), 7.64(d, 2H), 6.64(d, 2H), 4.47(d, 2H), 4.21(t,1H), 3.68-3.35(m, 38H), 3.07 (t, 4H), 2.32-2.11 (t, 6H),1.86(t, 1H).LCMS: MH⁺ 977, retention time 1.96 min.

LCMS Method: Column—YMC TRIART C18 (33×2.1 mm, 3u); (mobile phase: 95%[0.1% HCOOH in water] and 5% [0.1% HCOOH in CH3CN] held for 0.50 minthen to 1% [0.1% HCOOH in water] and 99% [0.1% HCOOH in CH3CN] in 3.0min, held this composition up to 4.00 min and finally back to initialcondition in 4.10 min, held for 4.50 min). Flow rate—1.0 ml/min.

Example 3 Synthesis of Nanoparticle Drug Conjugates (NDCs) Preparationof Nanoparticles

Aqueous synthesis methodology can be used for the preparation andfunctionalization of ultrasmall nanoparticles of the present disclosure.For example, methodology based on the procedures outlined in WO2016/179260 A1 and WO 2018/213851 A1 (the contents of which areincorporated herein by reference in their entireties) may be used.

For example, a fluorescent compound such as, but not limited to Cy5, canbe functionalized with a maleimide group, to provide amaleimide-functionalized fluorescent compound that has a net positivecharge. This can be conjugated with a thiol-silane, such as(3-mercaptopropyl)trimethoxysilane (MPTMS) to produce asilane-functionalized fluorescent compound such as Cy5-silane. Theconjugation may be performed in dimethyl sulfoxide (DMSO) in a gloveboxunder inert atmosphere overnight (16-24 hours) and at room temperature(18-25° C.).

On the following day, the next step of the synthesis can be performed ina suitable chamber, such as a glass flask, container, or reactor, andcan involve stirring deionized water with a pH of around 8.5-10.5 whichcan be achieved using an aqueous solution of ammonium hydroxide of pH7.5-8.5. A silica precursor, such as a tetraalkyl orthosilicate, e.g.,tetramethyl orthosilicate (TMOS), can then be added into the reactionchamber under vigorous stirring at room temperature, followed byimmediately adding the silane-functionalized fluorescent compound, e.g.,Cy5-silane. The reaction can be left stirring at room temperatureovernight (1-48 hours), to provide silica cores encapsulating thefluorescent compound, e.g., Cy5 dye.

The following day, a PEG-silane can be added into the reaction understirring at room temperature to coat the silica core with PEG molecules,and the reaction can be left stirring for 1-48 hours. This step may befollowed by heating between 75-85° C. for 1-48 hours. The reaction canthen be cooled down to room temperature and purified (e.g., includingsterile filtration to remove aggregates formed as side-product of thereaction, and bacteria if any present). Further functionalization of thenanoparticle may then be performed. Functionalization of Nanoparticles

A nanoparticle prepared using a method disclosed herein may be furtherfunctionalized, e.g., using a method outlined in FIG. 2 or 3 , or inScheme 6 below. For example, (3-cyclopentadienylpropyl)triethoxysilane(“diene-silane”) can be used to functionalize a nanoparticle (e.g.,C'Dot) with cyclopentadiene groups, then DBCO-PEG-maleimide can bereacted with the diene-functionalized nanoparticle to provide aDBCO-functionalized nanoparticle.

For example, Cy5-C'Dot (which may be prepared using a method describedherein) was diluted with deionized water to a desired concentration,typically between 15 to 30 μM, in a round-bottom flask with a stir bar.(3-Cyclopentadienylpropyl)triethoxysilane (cyclopentadiene) was firstdiluted 100× in DMSO and then added into the reaction with stirring, toreach a desired particle to cyclopentadiene molar ratio. After overnightreaction, 10> PBS was added into the reaction to achieve a finalconcentration of 1× PBS. Next, a DBCO-maleimide precursor (e.g.,DBCO-PEG4-maleimide) was dissolved in DMSO and added into the reactionto reach a desired particle to DBCO molar ratio. After mixing for about30 min to 1 hour, the reaction mixture was heated to 80° C. whilestirring overnight. The reaction solution was then concentrated andpurified using gel permeation chromatography (GPC) to obtain diene-basedDBCO-C'Dot.

The purification may be performed based on the principle of sizeseparation. Aggregates and free small molecules having molecular weightdifferent than that of the pegylated nanoparticles are separated usinggel permeation chromatography columns (GPC) or Tangential FlowFiltration (TFF) system. Two different membranes, 300 kDa, and 50 kDacut-off sizes were employed for the removal of large aggregates and freesmall molecules respectively. Both GPC and TFF systems can be used totransfer the aqueous medium to water, saline etc. Purified DBCO-C'Dot indeionized water can be sterile filtered again and the quality control(QC) steps can be performed, followed by storage in refrigerator at 2-8°C.

Without wishing to be bound by theory, it is believed that the neutralcharge of the cyclopentadiene groups averts hydrolysis of the amidebonds in the linkage, that can be accelerated by other types ofprecursors (e.g., when using amine-silanes instead of diene-silanes, theprimary amine groups can cause hydrolysis). Thus, the NDCs producedusing this method are highly stable (see, e.g., comparison in FIGS.33A-33B). Additionally, using diene-functionalized nanoparticles (e.g.,cyclopentadiene-functionalized nanoparticles) in the preparation of NDCSgreatly diminishes the self-condensation of silane during the reaction,and improves the stability, size homogeneity, reaction yield, and purityof the functionalized nanoparticles, relative to other methods (e.g.,using amine-silanes).

Preparation of Targeted NDCs

NDCs of the present disclosure comprising the nanoparticle (alsoreferred as C'Dot), targeting ligand (folic acid) and linker-drugconjugates can be prepared as outlined in the flow chart presented inFIG. 3 , and in Scheme 7 below. By adjusting the amount of targetingligand precursor used in the functionalization step, a desired number oftargeting ligands per nanoparticle can be achieved. For example,nanoparticles of the present disclosure may be functionalized to containabout 10 to about 20 folic acid moieties, e.g., about 10, about 11,about 12, about 13, about 14, or about 15 folic acid moieties.Similarly, by adjusting the amount of payload-linker conjugate precursorused in the functionalization step, a desired number of payload moietiesper nanoparticle can be achieved. For example, nanoparticles of thepresent disclosure may be functionalized to contain about 10 to about 40exatecan-linker moieties, e.g., about 20, about 21, about 22, about 23,about 24, or about 25 exatecan moieties.

Synthesis offolic-acid conjugated nanoparticle: DBCO-C'Dot (referred asC'Dot in FIG. 3 ) was diluted using deionized water to a concentrationof 15-45 μM. After the temperature of DBCO-C'Dot solution was around18-25° C., folate receptor (FR)-targeting ligand precursor such as,folic acid (FA) functionalized with an azide (compound 606 prepared inExample 2) was dissolved in DMSO (0.021 M) and was then added into thereaction with stirring at room temperature, providing a C'Dotfunctionalized with FA via the DBCO group on the surface. The reactionratio between DBCO-C'Dot and FA was kept from 1:5 to 1:30, and thesolution was stirred for 16-24 hours at temperature of 18-25° C.FR-targeting ligand addition is followed by sterile filtration,purification and QC testing to yield FA-C'Dot (referred as C'Dotintermediate in FIG. 3 ), and can be stored in a refrigerator at 2-8° C.FA-C'Dot comprises a portion of DBCO groups that are available forfurther click-reactions, e.g., with molecules with azide functionality.It will be understood that the folate-targeting ligand (e.g., folicacid) can be conjugated to the nanoparticle after conjugation with,e.g., a payload-linker conjugate.

The volume of the FR-targeting ligand conjugation reaction can rangefrom 5 mL to 30 L, and the concentration of DBCO-C'Dot can range from 15to 45 μM. The following parameters are given for a typical reactionvolume of 600 mL and a DBCO-C'Dot concentration of 25 μM. The ratio ofDBCO-C'Dot to FR-targeting ligands was precisely controlled to obtainthe desired number of FR-targeting ligands per particle, and typicallycan range from 1:5 to 1:30. For a typical ratio of 1:12,folate-PEG-azide was dissolved in DMSO to a concentration of 0.021 M,and 8.571 mL of the folate-PEG-azide/DMSO solution was added into thereaction. After stirring overnight at room temperature, the reactionmixture was either purified to obtain FA-C'Dot or continue directly tonext conjugation step if the purity of FA-C'Dot is no less than 95%. Theconversion rate of FR-targeting ligand is typically higher than 95%.

The number of folic acid groups attached onto each FA-C'Dot wascharacterized by UV-Vis, and a representative UV-Vis absorbance spectrumis shown in FIG. 4 . The number of DBCO groups on each C'Dot can becalculated using the extinction coefficient of C'Dot and DBCO groups

Synthesis ofFA-targeted NDC (or FA-CDC) comprising exatecan: FA-C'Dotswere diluted using deionized water to a concentration of 15-45 μM. Afterthe FA-C'Dot solution temperature reached around 18-25° C.,exatecan-linker conjugate precursor (e.g., compound 202 described inExample 1) cathepsin dissolved in DMSO (0.04 M) was added into thereaction under stirring at room temperature. This step functionalizedthe FA-C'Dot with the linker-drug conjugate via the available DBCOgroups on the surface. The reaction ratio between FA-C'Dot andlinker-drug conjugate was kept around 1:10-1:50 and the solution wasstirred for 16-24 hours. The addition of linker-drug conjugate wasfollowed by sterile filtration, and purification. FA-CDC (also referredas NDC) in deionized water is QC tested, and stored in refrigerator at2-8° C.

The volume of the cleavable exatecan conjugation reaction can range from5 mL to 30 L, and the concentration of FA-C'Dot can range from 15 to 45μM. The following parameters are given for a typical reaction volume of600 mL and a FA-C'Dot concentration of 25 μM. The ratio of FA-C'Dot tocleavable exatecan was precisely controlled to obtain the desired numberof cleavable exatecan per particle, and typically can range from 1:10 to1:60. For a typical ratio of 1:40, cleavable exatecan was dissolved inDMSO to a concentration of 0.04 M, and 15 mL of the cleavableexatecan/DMSO solution was added into the reaction. After stirringovernight at room temperature, the reaction mixture was purified toobtain FA-CDC.

The number of exatecan payloads attached onto each NDC, e.g., folic acid(FA)-functionalized drug-linker conjugated C'Dot (FA-CDC), may becharacterized by UV-Vis. A representative UV-Vis absorbance spectrum isshown in FIG. 5 . The number of exatecan payloads on each C'Dot can becalculated using the extinction coefficient of C'Dot and Exatecan at 360nm after the subtraction of the absorption of Folic Acid at the samewavelength.

As stated above, a nanoparticle may be functionalized with a folatereceptor targeting ligand and a payload-linker conjugate in any order(e.g., the protocol outlined above for functionalizing the nanoparticlewith exatecan may be carried out prior to the protocol for conjugatingfolic acid).

Particle Size Determination: The average diameter of NDCs can bemeasured by any suitable methods, such as, but not limited tofluorescence correlation spectroscopy (FCS) (FIG. 6 ) and gel permeationchromatography (GPC) (FIG. 7 ).

FCS detects the fluorescence fluctuation resulted from particlediffusion through the focal spot on the objective. Particle diffusioninformation is then extracted from the autocorrelation of signalintensity fluctuations, from which the average hydrodynamic particlesize can be obtained by fitting the autocorrelation curve using asingle-modal FCS correlation function. The average hydrodynamic diameterof NDC was about 6 nm to about 7 nm (FIG. 6 ).

GPC is a type of molecular sieving chromatography, where the separationmechanism is based on the size of the analyte (here NDC's). The elutiontime of NDC is compared to a series of proteins with varying molecularweight. The results suggest that the elution time of NDC's is comparableto that of protein standards with molecular weight between 158 kDa and44 kDa, consistent with the particle size average hydrodynamic size ofabout 6.4 nm (FIG. 7 ).

Purity Analysis: The purity of NDCs was analyzed using reverse phaseHPLC (RP-HPLC). RP-HPLC is coupled to a photodiode array detector, usinga commercially available Waters Xbridge Peptide BEH C18 column. RP-HPLCseparates molecules with different polarities and is suitable as ananalytical method for NDCs because of its ultrasmall sub-10 nm particlesize. Using RP-HPLC, the nanoparticles are well separated fromaggregates and other chemical moieties such as targeting ligands thatare non-covalently associated with the nanoparticles and degradedproducts. Different chemical moieties are identified based on theirelution time and unique UV/Vis spectra. The photodiode array detectorcollects UV-Vis spectra from 210 to 800 nm, and impurities of interestare measured at 330 nm. A representative chromatogram shown for the NDCsin FIG. 8 , suggests that the purity of NDCs of the present disclosureis higher than 99.0%. Example 4: Drug-Release Assays

NDCs of the present disclosure comprise a linker-payload conjugate,e.g., a protease-cleavable linker, such as cathepsin-B (Cat-B) cleavablelinker. Upon contact with a protease, the NDCs may release the payload(i.e., exatecan). The drug-releasing profile and the stability oflinker-drug conjugates on the nanoparticle were tested according to thefollowing protocols.

The NDCs were prepared using methods described in Example 3, and thenincubated under the desired releasing conditions for release kineticstests. The NDCs tested in the assay are provided in Table 2 below.

TABLE 2 Exemplary NDCs used in drug-release assays. NDC Exatecan-LinkerConjugate Structure B

C

D

Number of FA ligands per particle is between 12 and 22; Number oflinker-drug conjugates per particle is between 17 and 25. Eachpayload-linker is conjugated to the NDC via a DBCO moiety (preparedaccording to the protocol outlined in Example 3).

Exatecan exhibits an absorption maximum at a wavelength of around 360 nm(FIG. 9 ), and this signal can be used to trace the payloads inhigh-performance liquid chromatography (HPLC) for releasing andstability studies. The amount of released drugs vs non-released drugswas measured using reverse phase HPLC by analyzing the area under curve(AUC) (FIG. 10A and FIG. 10B).

General Method: A Waters Xbridge Peptide BEH C18 column with 4.6 mm×50mm dimensions, a particle size of 5 μm, and a pore size of 300 Å wasused (part number 186003622). Acetonitrile (VWR HiPerSolv Chromanorm,UHPLC Grade) was used as received without further preparation, 0.01%trifluoroacetic acid in deionized water was prepared by adding 1 mL oftrifluoroacetic acid (HPLC grade, Millipore-Sigma) into 999 mL 18.2MΩ·cm deionized water that was generated using an IQ7000 Milliporedeionized water system and passed through a 0.2 μm filter before use.The seal wash used for the system was composed of 90% 18.2 MΩ·cmdeionized water and 10% methanol (HPLC grade, VWR). The injection needlewas washed using a mixture of 25 vol % 18.2 MΩ·cm deionized water, 25vol % acetonitrile, 25 vol % methanol, and 25 vol % 2-propanol. Sampleswere prepared in a concentration range of 0.25 to 2 μM and the injectionvolume ranges from 60 μL to 10 μL, respectively. Higher sampleconcentration can be used if detector signal is low. Vials used for allinjections are fresh Waters Total Recovery vials with screw caps thathave pre-slit PTFE septa (part number 186000385C).

Before any sample injections were started, the PDA lamp was turned onand allowed to warm up for at least 30 minutes. The system and columnwere equilibrated with 95% 0.01% TFA in deionized water, 5% acetonitrilefor at least 10 minutes at a flow rate of 1.0 mL/min after the PDA lamphad warmed up. Two blank injections, with injection volumes of 10 μLcontaining only 18.2 MΩ·cm deionized water, were performed before theinjection of any samples for analysis. The gradient used began at 95%0.01% TFA in deionized water and 5% acetonitrile and linearly changed to15% 0.01% TFA in deionized water, 85% acetonitrile over 8 minutes.Acetonitrile composition was increased to 95% over an additional minuteand held at 95% for an additional 2 minutes to ensure that any stronglyretained compounds are eluted. The composition of the solvent was thenchanged back to the starting composition of the gradient over anadditional minute and allowed to equilibrate for 3 minutes beforeanother injection began. Between sample injections a blank injection wasrun to ensure that no carryover occurred.

For a typical cathepsin B (Cat-B) protease cleaving study, 2 μL, 0.33μg/μL of Cat-B (sigma Aldrich) was first added with 300 μL of activationbuffer (25 mM MES, 5 mM DTT, pH 5.0), forming 2.2 μg/mL of Cat-B. Themixture was kept at room temperature for 15 min before use. Afteractivation, 100 μL of 2 μM drug-nanoparticle-conjugate was mixed with100 μL of activated Cat-B. The mixture was then transferred to 37° C. Tomonitor the cleaving kinetics, at selected post-incubation time points(e.g., 2, 4, 24 h), 10 μL of mixture was sampled and injected in HPLC(TFA/acetonitrile). For the analysis of cleaving data, Empower 3ApexTrack integration was used to determine peak areas for all relevantcomponents.

The RP-HPLC chromatograph of three representative NDCs (NDC B, NDC C,and NDC D) at different time points after incubation with cathepsin-B isdepicted in FIGS. 11A-11C, respectively. The time for half of thepayloads to be released from each NDC, i.e., T_(1/2), under the specificexperimental condition was analyzed by fitting and is depicted in FIGS.12A-12C respectively. FIG. 12A depicts the T_(1/2) as 2.9 hours for NDCB. FIG. 12B depicts the T_(1/2) as 2.6 hours for NDC C. FIG. 12C depictsthe T_(1/2) as 1.4 hours for NDC D.

Stability Test: To assess the drug releasing profile and stability ofthe linker-drug conjugates under non-cleavage conditions, an exemplaryNDC was incubated in phosphate-buffered saline (PBS) buffer or animalserum at 37° C. The NDC was prepared according to Example 3, using theexatecan-linker conjugate precursor 202 from Example 1)

For a typical stability test in PBS buffer, 600 μL of PBS mixture(drug-nanoparticle-conjugate concentration was kept at 2 μM, while thevolume percentage of PBS was kept as 50%) was prepared and kept at 37°C. To monitor the stability of the linker-drug conjugates attached tonanoparticles, at selected post-incubation time points (e.g., 4, 24, 48and 72 h), 10 μL of mixture was sampled and injected in HPLC(TFA/acetonitrile). For the analysis of cleaving data, Empower 3ApexTrack integration is used to determine peak areas for all relevantcomponents.

For a typical stability test in plasma from varied species (e.g., mouse,rat, dog, monkey and human), 600 μL plasma mixture(drug-nanoparticle-conjugate concentration was kept at 2 μM, while thevolume percentage of plasma was kept as 62.5%) was prepared and kept at37° C. To monitor the stability of linker-drug conjugates, at selectedpost-incubation time points (e.g., 4, 24, 48 and 72 h), 80 μL of mixturewas first mixed with 80 μL of cold acetonitrile, and then went through30 min of centrifugation at 10,000 rpm. After removal of the proteins,60 μL of supernatant was carefully sampled and injected in HPLC. Forcathepsin-B-cleavable NDC, TFA/acetonitrile was used. For the analysisof stability data, Empower 3 ApexTrack integration was used to determinepeak areas for all relevant components.

The linker-payload conjugate of the NDC (as prepared in Example 3, usingthe exatecan-linker conjugate precursor 202 from Example 1) is stable,as 5% of less of the exatecan was released from the linker drugconjugate after 24 hours under non-cleavage conditions, i.e., whenmaintained in PBS, human serum, or mouse serum.

Example 5 In Vitro Flow Cytometry Cell Binding Study

Cell-binding activity of the NDCs disclosed herein was tested accordingto the following protocols. NDCs used were prepared according to Example3, using the exatecan-linker conjugate precursor 202 of Example 1. Theamount of folic acid per nanoparticle, and the amount of exatecan pernanoparticle, could be adjusted according to the protocol outlined inExample 3.

Cells and Cell Culture: Human KB cell line, SKOV-3 cells and TOV-112cell line were purchased from ATCC. I-GROV1, human ovarian carcinomacell line was purchased from EMD Millipore. Cells were maintained inFolic Acid free RPMI 1640 media/10% FBS, and 1% ofpenicillin/streptomycin, unless otherwise specified. Cancer cells werecultured in folic acid-free medium (RPMI1640, ThermoFisher, GIBCO) forat least one week before the study. Cell binding studies were performedby incubating 5×105 cells (total of 500 uL, 1 Million/mL) in cold PBS(with 1% of BSA) with FA-CDC prepared in Example 3 (concentration: 1 nM)for 60 min at 4° C. (n=3). After that, the cell suspension was stainedwith viability kit (LIVE/DEADTM Fixable Violet Dead Cell Stain Kit,Thermo Fisher) for 10-15 min. Then, cells were centrifuged (2000 rpm, 5min), washed (2-3 times) using cold PBS (with 1% of BSA) beforeresuspending in PBS (with 1% of BSA). Triplicate samples were analyzedon a LSRFortessa flow cytometer (BD Biosciences) (Cy5 channel, 633nm/647 nm, Live/dead cell stain, 405 nm). Results were processed usingFlowJo and Prism 7 software (GraphPad).

The competitive binding study (FIG. 13 ) was performed using the NDC ofExample 3. The active targeting of NDC can be fully blocked byincubating with the presence of 1 mM free Folic Acid.

The competitive binding study shows >40-fold enhancement in bindingcapability of the NDC when compared with free folic acid, demonstratingthe presence of a multivalent effect when conjugating multiple folicacid ligands on each ultrasmall C'Dot (FIG. 13 ).

These results demonstrate the advantages of conjugating multiple smalltumor-directing ligands on the surface of nanoparticle (C'Dots) forenhancing the targeting capability using the multivalent effect. Thefolate receptor targeting can be blocked by competitive binding of freefolic acid, such as by incubating with the presence of 1 mM free FolicAcid.

The flow cytometry shows comparable folate receptor targeting efficacyof two NDC formulations with varied folic acid ligand density, in KBcell line. The linker-exatecan conjugate precursor used to prepare theNDCs in this study is described in Example 1 (Compound 202). Theblocking group has 1 mM of free Folic Acid. (FIG. 14 ).

The results demonstrated dramatic increase (>300-fold of MFI) in folatereceptor-alpha active targeting when the folic acid ligand density wasincreased from zero to 12 (i.e., 12 folic acid molecules pernanoparticle), while little difference was observed upon furtherincreasing that density to 25 folic acid molecules per nanoparticle.

The flow cytometry shows comparable folate receptor targeting efficacyof three NDCs in KB cell line with varied drug per particle (DPR) (i.e.,number of exatecan molecules per nanoparticle). The blocking groupinvolved blocking receptors with 1 mM of free folic acid. The NDCs withdifferent ratios of exatecan per nanoparticle were prepared usingCompound 202 described in Example 1, and the results of the study areprovided in FIG. 15 . All FA-CDCs comprise between 12 and 22 folic acidmoieties. FA-CDCs with high drug-particle ratio (DPR) comprise between35 and 50 exatecan-linker conjugate groups. FA-CDCs with medium DPRcomprise between 17 and 25 exatecan-linker conjugate groups. FA-CDCswith low DPR have between 5 and 10 exatecan-linker conjugate groups.

These results together with the nearly unchanged FCS sizing changes ofthe three NDCs demonstrate the robust surface chemistry and maintainedfolate receptor targeting capability of NDCs disclosed herein, which issurprisingly not perturbed by altering the loading capacity of payloadand demonstrates a significant advantage of the NDCs disclosed hereinover other drug delivery platforms.

Pre-incubating NDCs in human plasma did not negatively affect folatereceptor targeting ability. This study was designed to test the possiblenegative impact of human plasma on the NDCs, such as the formation ofprotein corona. The formation of protein corona and its negative impacton the designed active targeting capability of drug delivery system hasbeen well documented in the literature. The results of this flowcytometry study are depicted in FIG. 16 , which show nearly unchangedfolate receptor targeting efficacy of NDCs at 1 nM, after pre-incubationwith varied amounts of human plasma for 24 hours. The NDCs were preparedaccording to the method of Example 3, using the exatecan-payloadconjugate precursor of Example 1 (Compound 202). The blocking groupinvolved blocking with 1 mM of free folic acid. This study clearlydemonstrated that the formation of a protein corona (if any) on the NDChad nearly no negative impact on the in vitro targeting capability ofthe NDCs.

Example 6 In Vitro Cell Viability Assay

The in vitro cytotoxicity of the NDCs disclosed herein were tested incancer cells. The cancer cells were cultured in folic acid-free medium(RPMI1640, ThermoFisher, GIBCO) for at least one week before the study.Cells were plated in opaque 96-well plates at a density of 3×10³ cellsper well (total of 90 mL) and allowed to attach overnight. The followingday, cells were treated with NDC (prepared according to Example 3) at aconcentration range of 0-50 nM (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5,1, 5, 10, 50 nM) by adding 10 mL of 10x stock FA-CDC solution.

Cells were treated for a pre-defined exposure time (depending on thestudy design, e.g., 4-6 hours, or 7 days). In the case ofshort-exposure-time viability study, cancer cells in each wells werewashed with 100 mL PBS and refreshed with 100 mL of cell medium. Afterwashing, the plates were returned back to 37° C. incubator for 7 daysbefore the viability assay. In the case of 7-day-exposure-time viabilitystudy, no additional washing step was performed. After 7 days, the cellviability was assessed using the CellTiter-Glo2.0 assay (Promega)according to manufacturer's instructions. Data for both viability andproliferation were plotted using Prism7 software (GraphPad).Representative cell viability results of six FA-CDCs with similarsurface density of Folic Acid targeting ligands and drug linkers ispresented in provided in Table 3.

TABLE 3 Representative cell viability results of NDCs with similarsurface density of folic acid targeting ligands and linker-drugconjugates. IC₅₀ in IC₅₀ in KB cell SKOV-3 cell Payload-Linker Conjugateline (nM) line (nM)

0.2-5.2 10.7

0.5-1.0 17.9

0.7-7.2 n.t.

17.5 n.t.

0.2-2.2 0.4

5.2 n.t.

72.2 n.t.

42.7 n.t.

0.6 0.9

49.4 n.t.

0.3 0.13Number of FA ligands per particle is between 12 and 22; Number oflinker-drug conjugates per particle is between 17 and 25. Eachpayload-linker is conjugated to the NDC via a DBCO moiety (preparedaccording to the protocol outlined in Example 3).

Example 7 Two-Dimensional (2D) Confocal Imaging of NDC in Cancer Cells

A 2D confocal imaging study was carried out to determine the targetingof cells with varying levels of folate-receptor availability, using twoexemplary NDCs. The cells with high folate-receptor expression (denoted++++) were KB cells. The cells with no FR expression (denoted (−) wereTOV-112D cell line. FR-blocked cells were also used.

KB cells were maintained in folic acid free RPMI 1640 media with 10%FBS, 1% penicillin/streptomycin. TOV-112D cells were maintained in 1:1mixture of MCDB 105 medium containing a final concentration of 1.5 g/Lsodium bicarbonate and Medium 199 containing a final concentration of2.2 g/L sodium bicarbonate, supplemented with 15% FBS and 1%penicillin/streptomycin. Cells were trypsinized and seeded in 8-wellLab-Tek chambered coverglass, at 1.0×105 cells per well, and culturedovernight to allow for attachment.

The NDCs were prepared according to Example 3 and are displayed below inTable 4. NDC D was prepared using the linker-payload conjugate (202)described in Example 1.

TABLE 4 Exemplary NDCs used in 2D Confocal Imaging Assay. NDCExatecan-Linker Conjugate B

D

Before incubation with NDC, cells were washed once with folic acid freeRPMI 1640 media. The NDC was added into folic acid free RPMI 1640 mediato final concentration of 50 nM. For blocking conditions, folic acid (20mM stock dissolved in 0.1 M NaOH) was added to final concentration of0.1 mM and co-incubated with NDC. Cells were incubated with NDC at 37°C. for either 1 hours or 24 hours. After incubation, cells were washedthree times. To stain lysosomes, LysoTracker Green DND-26 (Thermo FisherCat. L7526, ex/em504/511 nm) was added to final concentration of 100 nMin folic acid free RPMI 1640 media with 10% FBS, 1% P/S, and incubatedat 37° C. for 45 min. Cells were washed once to remove remaininglysotracker dyes. To stain nuclei, Hoechst 33342 solution (Thermo FisherCat.62249, 20 mM) was diluted 1:4000 in Folic Acid free RPMI 1640 mediawith 10% FBS, 1% P/S, and incubated at 37° C. for 10 min. Cells werewashed once, and media was exchanged to phenol red free RPMI 1640 mediafor confocal imaging using Nikon spinning disk confocal microscope, 60xobjective, 405 nm, 488 nm, 640 nm laser lines, exposure time 100 ms for405 channel, 500 ms for 488 channel, and 600 ms for 640 channel.

Results from confocal microscope imaging of NDC in KB (++++) andTOV-112D(−) cell lines at 1 hour time point showed that NDC were mainlypresent at the cell membrane of KB cells, which express high level offolate receptors, but not in blocking conditions or folate negative cellline TOV-112D, suggesting specific binding of NDC to folate receptors.After 24 hours, membrane bound NDC were internalized and the amount ofinternalized NDC significantly increased as compared with 1 hour timepoint. The internalized NDC were localized in acidic organelles stainedby LysoTracker, indicating that the trafficking of NDC occurred thoughthe endo-lysosomal pathway. The effect of serum on the bindingcapability of NDC was also investigated by pre-incubating NDCs in mediasupplemented with 10% FBS overnight, prior to incubating them withcells, and no significant difference was observed (data not shown),suggesting that the presence of serum had no impact on the bindingcapability of NDCs.

These results of confocal microscopy of NDC B are provided in FIG. 17 ,and results for NDC D are provided in FIG. 32 . These imagesdemonstrated the highly specific active targeting and lysosometrafficking of the NDCs of the present disclosure, indicating that oncethe FA-targeting NDCs bind to cells they become internalized in folatereceptor positive cell lines, where the exatecan payload may be cleaved(e.g., by cathepsin-B) to release free exatecan in the cancerous cell.

Example 8 Confocal Imaging of FA-CDC in 3D Tumor Spheroid Model in KBCells

A 3D tumor spheroid model assay was conducted to determine the tumorpenetration of the NDCs disclosed herein. The assay compared anexemplary NDC (prepared according to Example 3, using exatecan-linkerconjugate precursor 202 of Example 1), with a payload-free FA-targetingnanoparticle (also prepared according to Example 3, with only the FAprecursor and without exatecan-payload conjugate precursor); a folatereceptor (FR)-targeting ADC; and the corresponding payload-freeFR-targeting antibody. The FR-targeting antibody was prepared based uponthe published sequence of mirvetuximab (provided in U.S. Pat. No.9,637,547 as huMovl9; the contents of which are incorporated herein byreference in its entirety). The ADC was prepared with the same antibodyand was conjugated to the maytansinoid drug DM4 (created by SyngeneInternational Ltd.) via a 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyricacid (sSPDB) linker (based on the linker used in U.S. Patent No.9,637,547). The ADC and antibody were each conjugated with Cy5 organicdye, by reaction with Cy5-NHS ester, and were purified by a PD-10column.

Corning ultra-low attachment surface 96-well spheroid microplates wereutilized in seeding KB cells for having KB spheroids with cell density10,000/well. Single-cell suspensions were generated from trypsinizedmonolayers and diluted to 100,000 cells/mL using RPMI medium (folic acidfree). 100 mL of cell suspension were dispensed into each well of amicroplate. The plate was kept in an incubator for 24 hours for cellsforming spheroids. KB cell spheroids can be easily observed bymicroscope with 10× objective.

3D KB spheroids formed after overnight culturing in an ultra-low 96-wellmicroplates. NDC (prepared according to Example 3, using exatecan-linkerconjugate precursor 202 of Example 1), folate-targeted nanoparticles(“FA-C'Dot”), FR-targeted ADC, or payload-free FR-targeted-antibody wereadded into wells (n=3) with 50 nM final concentration and incubated for4 hours at 37° C. Each treated KB spheroid and control spheroid werewashed with PBS for three times and then carefully transferred to aglass bottom 96-well plate (Cellvis) for observation by Nikon A1R-STEDconfocal microscope, using laser line 640 nm, 20× objective. Z-stackswere acquired by taking 2-dimensional images each separated by 1 μm inthe Z-direction.

Results from the Z-stack confocal microscope imaging of KB tumorspheroid treated with the NDC, FA-C'Dot, FR-targeted ADC, andpayload-free FR-targeted antibody is depicted in FIG. 18 . The resultsshow that the penetration and well diffusion of NDC and FA-C'Dotsthroughout the whole >800 mm of tumor spheroids. In contrast, labeledantibody and ADC merely accumulated around, but not inside of, the tumorspheroids. The ability of the NDCs disclosed herein to achieve efficienttumor penetration is highly advantageous, and shows significantimprovement compared to conventional drug delivery platforms.

Example 9 ⁸⁹Zr Radiolabeling of DFO-FA-CDC and In Vivo Static PET/CT andBiodistribution Studies

A radiolabeling assay was conducted to determine the in vivobiodistribution of the folate receptor-targeting NDCs of the presentdisclosure. The NDCs used for the assay were conjugated with thechelator desferrioxamine (DFO) and then bound with a radionuclide(⁸⁹Zr).

For a typical ⁸⁹Zr labeling, about 1 nmol of DFO-conjugated NDC weremixed with 1 mCi of ⁸⁹Zr-oxalate (produced and provided by University ofWisconsin-Madison Cyclotron group) in HEPES buffer (pH 8) at 37° C. for60 min; final labeling pH was kept at 7-7.5. The labeling yield could bemonitored by using radio instant thin-layer chromatography (iTLC). Anethylenediaminetetraacetic acid (EDTA) challenge procedure was thenintroduced to remove any nonspecifically bound ⁸⁹Zr from the particlesurface. As-synthesized labeled NDC (⁸⁹Zr-DFO-FA-CDC) were then purifiedby using a PD-10 column. The final radiochemical purity was quantifiedby using iTLC.

For PET/CT imaging, healthy nude mice (n=3) were i.v.-injected with200-300 μCi (7.4-11.1 MBq) ⁸⁹Zr-DFO-FA-CDC. Approximately 5 min prior tothe acquisition of PET/CT images, mice were anesthetized by inhalationof 2% isoflurane/oxygen gas mixture and placed on the scanner bed;anesthesia was maintained using 1% isoflurane/gas mixture. PET/CTimaging was performed in a small-animal PET/CT scanner (InveonmicroPET/microCT) at 1-2, 24, 48, and 72 h post-injection. An energywindow of 350-700 keV and a coincidence timing window of 6 ns were used.Data were sorted into 2D histograms by Fourier rebinning, and transverseimages were reconstructed by filtered back-projection into a 128×128×63(0.72×0.72×1.3 mm3) matrix. The PET/CT imaging data were normalized tocorrect for nonuniformity of response, dead-time count losses, positronbranching ratio, and physical decay to the time of injection; noattenuation, scatter, or partial-volume averaging corrections wereapplied. The counting rates in the reconstructed images were convertedto activity concentrations (percentage injected dose per gram of tissue,% ID/g) by use of a system calibration factor derived from the imagingof a mouse-sized water-equivalent phantom containing ⁸⁹Zr.Region-of-interest (ROI) analyses of the PET data were performed usingIRW software. At 72 h post-injection, organs from each individual mousewere collected, wet-weighted and gamma counted (Automatic Wizard2γ-Counter, PerkinElmer). The uptake of ⁸⁹Zr-DFO-FA-CDC was presented as% ID/g (mean±SD).

The NDCs of the present disclosure enable precise tumor targeting, deeptumor penetration and high tumor killing efficacy. The NDCs can becleared rapidly and efficiently from the body, which reduces thepotential for off-target toxicities and results in an improved safetyprofile. The NDCs disclosed herein (comprising targeting ligands (folicacid) and payload (exatecan)) can be administered to a subject andcirculate through the blood stream, target the cancer (e.g., tumor),diffuse, penetrate, internalize, and cleave the exatecan payload,killing the cancer cells.

In this study, the renal clearance and biodistribution pattern of FA-CDCwere tested. As shown in FIG. 19A, after the intravenous injection, the⁸⁹Zr-DFO-FA-CDC circulated in the blood stream of healthy nude mouse, asindicated by the high radioactive signal from the heart and artery.Dominant radioactive signal can also be seen from the mouse bladder,demonstrate the renal clearance of the NDC. After 24 h, the majority ofthe injected ⁸⁹Zr-DFO-FA-CDC was cleared out of the mouse body. Thechanges in biodistribution pattern at 2 hours and 24 hourspost-injection is also shown in FIG. 19B. As expected, the NDC cancirculate in the blood stream with a dominant renal clearance pathway,whilst avoiding clearance by the mononuclear phagocytic system (MPS)(i.e., liver and spleen).

Example 10 Human KB Tumor Model and In Vivo Efficacy Study

The in vivo efficacy of the NDC was carried out using a human KB tumormouse model. The assay compared the NDC prepared according to Example 3using the exatecan-payload conjugate precursor 202 (Example 1; herelabeled D, and shown in FIG. 20D, with the NDCs indicated in Table 5below (NDCs A-C and E-F). Each NDC was compared to a control and freeexatecan, and NDCs E and F were compared to free exatecan and irinotecan(CPT-11).

TABLE 5 Exemplary NDCs used in the in vivo efficacy study NDCPayload-Linker Conjugate A

B

C

D

E

F

Number of FA ligands per particle is between 12 and 22; Number oflinker-drug conjugates per particle is between 17 and 25. Eachpayload-linker is conjugated to the NDC via a DBCO moiety (preparedaccording to the protocol outlined in Example 3).

Human KB cell line was purchased from ATCC and maintained in folic acidfree RPMI 1640 media/10% FBS, and 1% of penicillin/streptomycin, unlessotherwise specified. Once the KB cells were cultured to reach anadequate cell count, the cell viability was confirmed by a hemocytometerand trypan blue staining assay. For subcutaneous implantation, eachmouse was injected with KB cells at a density of 2×106 cells/mice at 0.1mL Matrigel/cell dilution volume per injection on the left lower flankof the thigh. Once a subcutaneous tumor volume has reached a palpablesize of 75 to 150 mm³ in a required number of mice for this study, themice was randomized and assigned to each treatment cohort resulting withcomparable tumor volume statistics. Following randomization and studycohort assignment, each dose cohort was treated according to the routesof administration, dosage and schedule.

Two dose levels of each of NDCs B-D were used in the efficacy study(only one dose level for NDCs A, E and F). Tumor volume measurementswere performed using a calibrated caliper every second day during thedose treatment period, followed by twice weekly measurements during therecovery period of the in-life phase, and tumor volumes were determinedusing the formula length (mm) x width (mm) x width (mm) x 0.50. Bodyweight measurements were performed every second day during the dosetreatment period, followed by twice weekly measurements during therecovery period of the in-life phase. Mice were euthanized when the endpoints of the study reached 1000 mm³. Tumors were harvested and tumorsize was measured. Tumor were surgically excised and snap-frozen forstorage at −80° C. until future analysis.

FIGS. 20A-20F depicts the in vivo tumor growth inhibition studies of thesix folate receptor-targeting NDCs in KB tumor-bearing mice (n=7). Thetumor growth charts depicted for the in vivo efficacy study shows aclear response of tumor growth inhibition in mice treated with the NDCprepared according to Example 3 using the exatecan-payload conjugateprecursor 202 (from Example 1), which is shown in FIG. 20D. Similarly,growth inhibition was observed in NDC A (FIG. 20A), NDC B (FIG. 20B),and NDC C (FIG. 20C). In contrast, mice treated with NDC E (FIG. 20E),and NDC F (FIG. 20F) showed no significant inhibition in tumor growth.Doses for the NDCs are provided in FIGS. 20A-20F. Clear response oftumor growth inhibition was observed in mice treated with NDCs A-D.Control group mice received normal saline follow the same Q3DX3 doseregimen.

Example 11 Activity of NDCs in Drug-Resistant Cell Lines

An assay was carried out using the NDCs disclosed herein to determinetheir activity in drug-resistant cancer cells (specifically,irinotecan-resistant KB cells and extecan-resistant KB cells). The NDCsused in this assay were prepared according to Example 3, using theexatecan-linker conjugate precursor 202 (from Example 1).

Development of TOP1 Inhibitor-Resistant Folate Receptor Alpha PositiveCancer Cells.

Naïve human KB cell line were purchased from ATCC and maintained infolic acid free RPMI 1640 media/10% FBS, and 1% ofpenicillin/streptomycin. To develop the TOP1 inhibitor resistant KBcells, the cells in flask (50-60% confluence) were repeatedly treatedwith increasing concentration of exatecan, topotecan, SN-38 oririnotecan for over 4 months. The starting TOP1 inhibitor treatmentconcentration was close to the KB cell's IC₉₀ values. After eachtreatment, the cells were carefully washed with fresh RPMI 1640 mediaand left to proliferate for an additional 2-3 days until reaching 50-60%confluence. The next round of TOP1 inhibitor treatment was started with2-10× higher TOP1 inhibitor concentration.

Resistant Factor and IC₅₀ Assay.

Both naïve and TOP1 inhibitor resistant KB cell were cultured in folicacid-free medium (RPMI1640, ThermoFisher, GIBCO). Cells were plated inopaque 96-well plates at a density of 3×10³ cells per well (total of 90μL) and allowed to attach overnight. The following day, cells weretreated with selected TOP1 inhibitors (e.g., free exatecan) or NDC atsuitable concentration ranges. After exposing the TOP1 inhibitors withboth types of cells for the same period of time, the cell viability wasassessed using the CellTiter-Glo2.0 assay (Promega) according tomanufacturer's instructions. Data for both viability and proliferationwere plotted using Prism? software (GraphPad). The resistant factor canbe calculated by using the following eauation:

${{Resistant}{Factor}} = \frac{{IC}_{50}{of}{resistant}{KB}{cell}}{{IC}_{50}{of}{naive}{KB}{cell}}$

Irinotecan-Resistant KB Cell Line and Potency Test of NDC

FIG. 21A shows the IC₅₀ curves of irinotecan in both naïve and resistantKB cells, which demonstrates the successful development of 5×irinotecan-resistant KB cells, where IC₅₀ free irinotecan inirinotecan-resistant KB cells was 3,618 nM, compared to 668 nM in naïvecells. FIG. 21B provides the IC₅₀ curves of the NDC (FA-CDC) (preparedaccording to Example 3, using the exatecan-linker conjugate precursor202 of Example 1) in the naïve KB cells (IC₅₀=0.27 nM) and resistant KBcells (IC₅₀=0.26 nM), indicating the NDC has a high potency that isuniform across both naïve KB cells and TOP1 inhibitor-resistant KBcells.

Exatecan-Resistant KB Cell Line and Potency Test of NDC

FIG. 22A shows the IC₅₀ curves of exatecan in both naïve and resistantKB cells, which demonstrates the successful development of >8×exatecan-resistant KB cells, where IC₅₀ of exatecan in regular KB cellswas 2 nM, compared with 4 nM in KB cells pretreated 4× with exatecan,and 16.9 nM in KB cells pretreated 7× with exatecan. FIG. 22B shows theIC₅₀ curves of the NDC (FA-CDC) (prepared according to Example 3, usingthe exatecan-linker conjugate precursor 202 of Example 1) in both naïveand resistant KB cells (4× or 7× pretreatment), where the IC₅₀ of theFA-CDC was 0.27 nM, 0.28 nM, and 0.30 nM, respectively. The resultsindicated the NDC possesses high potency uniformly in both the naïve andresistant KB cells.

Example 12 Activity of NDCs in Cancer Cells with Varied Folate ReceptorExpression Levels

An assay was conducted to determine the cytotoxicity of exemplary NDCs(FA-CDCs), with varying levels of drug-to-particle ratio, in differentFR-alpha overexpressing cancer cell lines, compared to non-conjugatedexatecan. The NDCs were prepared according to Example 3, using thepayload-linker conjugate precursor 202, of Example 1. The NDCs (FA-CDCs)tested had a drug-to-particle ratio (DPR) of 43, 20, 8, and 1 (i.e., 43,20, 8, and 1 exatecan-linker groups per nanoparticle).

Cancer cells with varied FR alpha expression levels (KB (++++),IGROV-1(++), SK-OV-3(++), HCC827(++), A549(−), and BT549(−)) werecultured in folic acid-free medium (RPMI1640, ThermoFisher, GIBCO) forat least one week before the study. Assays for 7-day exposure and 6-hourexposure were both conducted.

Cells were plated in opaque 96-well plates at a density of 3×103 cellsper well (total of 90 μL) and allowed to attach overnight. The followingday, cells were treated with NDC with varied drug-to-particle ratio(DPR) at a concentration ranging from 0-50 nM (0, 0.001, 0.005, 0.01,0.05, 0.1, 0.5, 1, 5, 10, 50 nM) by adding 10 μL of 10× stock compounds.

For the 6-hour exposure viability study cells were treated for 6 hoursand washed (3×) with 100 μL PBS. 100 μL of fresh cell medium was thenadded to each well and the plate was incubated for an additional 7 daysat 37° C. before performing the CellTiter-Glo® cytotoxic assay (Promega)according to manufacturer's instructions. The results of the 7-dayexposure assay are presented in FIG. 23 , which demonstrate that the NDCwas highly potent across all cell lines, despite differing levels of FRexpression in the cells.

For the 7-day exposure viability study, the cells were incubated withcompounds for the entire 7-day period, followed by the CellTiter-Glo®cytotoxic assay. Data for half maximal inhibitory concentration (IC50)was plotted using Prism? software (GraphPad). The results of the 6-hourexposure assay are presented in FIG. 23 , which demonstrate that the NDCwas highly potent across all cell lines, despite differing levels of FRexpression in the cells.

Example 13 Cytotoxicity of NDCs in Patient-Derived Pt-Resistant TumorCell Lines

An assay was conducted to establish the cytotoxicity of an exemplary NDC(prepared according to Example 1, using the exatecan-linker conjugateprecursor Compound 202 from Example 1) in various patient derived tumorcell lines that are Pt-resistant, with comparison to non-conjugatedexatecan. Cell lines were obtained from ovarian cancer, non-small celllung cancer (NSCLC), breast cancer (both HR+, HER2+; and HR-, HER2+; andtriple negative breast cancer (TNBC)), endometrial cancer, and head andneck (H&N) cancers. The results of the assay are provided in FIG. 24 .

The cytotoxic efficacy was determined by KIYATEC using the KIYA-PREDICT™assay. The FRa immunohistochemistry (ICH) scoring of tumor tissue fromplatinum-resistant ovarian, endometrium, non-small cell lung, breast,triple-negative breast, head & neck cancer patients were conducted byXenoSTART by using the Biocare Medical FRa IHC Assay Kit (cat#BRI4006KAA), following the manufacturer's protocol. A total of 28 PDXmodels from different indications were selected based on the IHC scoresand provided to KIYATEC for the KIYA-PREDICTTM assay. Briefly,cryopreserved PDX tumors were thawed and enzymatically dissociated tosingle cells, and plated into 384-well spheroid microplates (Corning).Flow cytometry was also performed to assess the FRalevels amongdifferent PDX models. Following the 24 hours of spheroid formation, NDCor controls were added at the designed concentration range and incubatedfor 7 days. After that, the cell viability was measured byCellTiter-Glo® 3D (Promega). The data was analyzed in Microsoft Exceland GraphPad Prism.

Example 14 In Vitro and In Vivo Efficacy of an Exemplary NDC inPediatric Acute Myeloid Leukemia Models

Assays were carried out to establish the in vitro and in vivo efficacyof an exemplary NDC (prepared according to the protocol in Example 3,using the exatecan-linker conjugate precursor 202 from Example 1) infolate-receptor alpha-positive pediatric acute myeloid leukemia models.

In Vitro Flow Cytometry Cell Binding Study

Cancer cells (IGROV-1 and AML MV4;11 cell lines) were cultured in folicacid-free medium (RPMI1640, ThermoFisher, GIBCO) for at least one weekbefore the study. Cell binding studies were performed by incubating5×10⁵ cells (total of 500 4, 1 million/mL) in cold phosphate-bufferedsaline (PBS) (with 1% of bovine serum albumin (BSA)) with the exemplaryNDC or with anti-FR alpha phycoerythrin (PE)-conjugated antibodies(anti-FR alpha antibody-PE) (concentration: 10 nM) for 60 min at 4° C.(n=3). A non-targeted CDC and isotype antibody-PE were used as negativecontrols for the exemplary NDC and anti-FR alpha antibody-PE,respectively. The cell suspension was then stained with viability kit(LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit, Thermo Fisher) for 10-15min. The cells were next centrifuged (2000 revolutions per minute, 5min), washed (2-3 times) using cold PBS (with 1% of BSA) beforeresuspending in PBS (with 1% of BSA). Triplicate samples were analyzedon a LSRFortessa flow cytometer (BD Biosciences) (Cy5 channel, 633nm/647 nm, Live/dead cell stain, 405 nm). Results were processed usingFlowJo and Prism 7 software (GraphPad).

The flow cytometry histograms of the exemplary NDC and anti-FR alphaantibody-PE compared with the respective negative controls (non-targetedNDC or isotype antibody-PE) are shown in FIGS. 25A-25D. The flow studydemonstrates the specific FR alpha targeting capability of the exemplaryNDC to both the IGROV-1 (FR alpha positive human ovarian cancer) and theAML MV4;11 cell lines.

In Vitro CellTiter-Glo® Cytotoxic Assay

Cancer cells (IGROV-1 and AML MV4;11 cell lines) were cultured in folicacid-free medium (RPMI1640, ThermoFisher, GIBCO) for at least one weekbefore the study. Cells were plated in opaque 96-well plates at adensity of 3×10³ cells per well (total of 904) and allowed to attachovernight. The following day, cells were treated with the exemplary NDCat a concentration ranging from 0-100 nM, by adding 10 μL of 10× stockNDC solution. For the shorter exposure viability study, cells weretreated for 4 hours and washed (3×) with 100 μL PBS. 100 μL of freshcell medium without the NDC was then added to each well and the platewas incubated for an additional 5 days at 37° C. before performing theCellTiter-Glo® cytotoxic assay (Promega) according to manufacturer'sinstructions. Data for half maximal inhibitory concentration (IC₅₀) wasplotted using Prism7 software (GraphPad).

The in vitro specific cytotoxic activity of the exemplary NDC in FRalpha positive human ovarian cancer and MV4; 11 AML cell lines isdisplayed in FIGS. 26A-26B. Cells were treated with the exemplary NDC atthe indicated concentrations, incubated at 37° C. for 4 hours, washed,and returned to the incubator for an additional 5 days, beforeperforming the CellTiter-Glo® cytotoxic assay.

CBFA2T3-GLIS2 Fusion Positive AML Cell Line-Derived Xenograft Models

In vivo anti-tumor killing activity of the exemplary NDC was assessed incell line-derived xenograft (CDX) models. NOD scid gamma (NSG) mice werefed with folate free chow for 1 week prior to injection with AML celllines. Then 1-5 million fusion-positive cell lines (M07e, WSU-AML) andengineered cells (MV4;11 FOLR+) transduced with Luciferase reporter weretransplanted into the NSG mice via tail-vein injections. Leukemia burdenand response to treatments was monitored using non-invasivebioluminescent imaging (from both the front and the back of the mouse),and flow cytometry analysis of mouse peripheral blood drawn bysubmandibular bleeds was carried out bi-weekly, starting from the firstweek of CDC treatment. Mice were monitored for disease symptoms(including tachypnea, hunchback, persistent weight loss, fatigue, andhind-limb paralysis). Mice from the saline control group (Cohort 1) wereeuthanized due to the high AML burden on Day 44 post-leukemia injection(tissues including blood, bone marrow, thymus, liver, lungs and spleenwere harvested at necropsy and analyzed for the presence of leukemiacells). Mice from the treatment groups (Cohorts 2-4) continued toreceive weekly bioluminescent imaging and bodyweight monitoring. Anillustration of the timeline for mice preparation, treatment, andimaging is provided in FIG. 31 .

All the mice were randomized prior to dosing and weighed to provide thecorrect designed dose based on Table 6 below. Leukemia burden andresponse to treatments was monitored weekly using non-invasivebioluminescent imaging. Bodyweight was measured every other day. Themice were terminated if their weight loss was over 20%.

TABLE 6 Dose design (n = 5 per group) Clinical Dose IV Dose ObservationsMaterial (mg/kg of volume and Study End Cohort Administered Study PhaseExatecan) Regimen (mL/kg) Points 1 Normal saline Vehicle control n/apQ3D × 3 10 Every other day 2 NDC escalation 0.33 Q3D × 6 10 body weight(BW) 3 NDC escalation 0.50 Q3D × 3 10 End point: BW 4 NDC escalation0.65 Q3D × 3 10 loss >20%

FIG. 27 provides the bodyweight change of AML mice treated with normalsaline and the exemplary NDC at the three dose levels indicated in Table6. The normal saline group (Cohort 1) showed a bodyweight loss within20%, mainly due to the leukemia burden. In the 0.33 mg/kg (Q3Dx6) dosegroup (Cohort 2), 4 of 5 mice tolerated the NDC well (<20% loss), andbodyweight was gained after 6 doses; while the remaining mouseshowed >20% bodyweight loss after the 5^(th) dose, and more bodyweightloss after the 6^(th) dose. In the 0.50 mg/kg (Q3Dx3) dose group (Cohort3), all 5 mice tolerated the NDC well (<20% loss), and bodyweight wasgained after 3 doses. In the 0.65 mg/kg (Q3Dx3) group (Cohort 4), 2 of 5mice tolerated the NDC well (<20% loss), and bodyweight was gained after3 doses, while 3 of 5 mice showed >20% bodyweight loss after the 3r^(d)dose.

FIG. 28 provides the in vivo bioluminescence images (BLI) obtained fromthe AML mice treated with normal saline or the exemplary NDC at eachdose regimen (i.e., Cohorts 1-4 from Table 6). Quantitative in vivobioluminescence imaging analysis of Cohorts 1-4 (i.e., AML mice treatedwith normal saline or the exemplary NDC at each dose regimen outlined inTable 6) is provided in FIG. 29 . In the normal saline group (Cohort 1),the leukemia burden continued to progress, with the average whole-bodyBLI signal increasing >90 fold in 34 days, while a quick anddose-dependent suppression of the leukemia burden was achieved in all 3treatment groups (Cohorts 2-4). The 0.5 mg/kg (Q3Dx3) dose group (Cohort3) showed 11-fold less leukemia burden on Day 34 when compared withburden on Day 1 post-leukemia injection. When comparing the 0.33 mg/kg(Q3Dx6) dose group (Cohort 2) with the 0.65 mg/kg (Q3Dx3) dose group(Cohort 4), 0.33 mg/kg was tolerated better with a slightly betterresponse. Taken together, these data indicate the exemplary NDCsuccessfully suppressed the leukemia burden in the FR alpha positive AMLmice, and showed quick and dose-dependent response.

FIG. 30 provides a graph illustrating the results of bone marrowaspiration of Cohorts 1-4 (i.e., the mice treated with normal saline orthe exemplary NDC at each dose group indicated in Table 6) on Day 42post-leukemia injection. Leukemia was detected in the group of micetreated with normal saline (Cohort 1), while no detectable leukemiaburden could be observed in any of the mice from the treatment groups(Cohorts 2-4).

Example 15 Stability of Linker Derived from Diene

In order to determine the stability of NDCs disclosed herein preparedusing a diene-based functionalization approach, the stability of NDCprepared using a diene based functionalization approach were compared toNDC prepared using an amine-based functionalization approach.

The NDCs were incubated in 0.9% saline, PBS, human plasma (10%), andmouse plasma (10%) at 37° C. in a shaking dry bath for different timeperiods. Prior to analysis, plasma proteins in the samples were removedby precipitation, through addition of an equivalent volume of coldacetonitrile, followed by centrifugation at 10000 rpm in an Eppendorf5425 microcentrifuge. Following centrifugation, the clear supernatantwas transferred from the centrifuge tube into a clear total recoveryHPLC vial. The supernatant free of any visible aggregation was dilutedwith an equivalent volume of deionized water to adjust the sample matrixto match the starting conditions of the HPLC separation and avoid lossof sensitivity. The purity and impurity of each sample is thenquantified by RP-HPLC.

The targeted-NDCs produced using the methods described in Example 3,using the diene-silane precursor exhibited high stability in mouse andhuman plasma, and showed significant stability improvement, relative tocorresponding NDCs produced using an amine-silane precursor (see FIGS.33A and 33B)). In the NDCs prepared using the diene-silane precursor,more than 95% of exatecan drugs remain on the NDCs for up to 7 days inmouse and human plasma, obtained by the UV-Vis spectra of the NDC peaksin RP-HPLC chromatograms. Meanwhile, an independent RP-HPLC assaymonitoring free exatecan suggested that the released exatecan was belowdetection limit of RP-HPLC, i.e., 0.02%, and the absence of non-desiredfree drug further demonstrates their high plasma stability. Thetargeted-NDCs also exhibited high storage stability at 4° C. in 0.9%saline. Their purity, size distribution, and hydrodynamic diameter werecharacterized by RP-HPLC, SEC and FCS respectively, and remainedunchanged over 6 months under storage condition. Such high storagestability is another key parameter important for both clinicaltranslation and commercial manufacture.

Example 16 Pharmacokinetics and Toxicology Study

The pharmacokinetics and toxicology of an exemplary NDC were assessed ina rat model and in a dog model. The NDC used in this study was preparedaccording to Example 3, using the exatecan-linker conjugate precursorcompound 202 of Example 1. As demonstrated in the above examples, thisexemplary NDC is highly stable in plasma and elicits antitumor efficacyin a variety of cell line and PDX-derived tumor models both in vitro andin vivo.

In 15-day repeat dose toxicology and toxicokinetic (TK) studiesperformed in Wistar Han rats and Beagle dogs, the NDC was tolerated atup to 0.87 mg/kg/day in rats and 0.174 mg/kg/day in dogs based uponconjugated exatecan concentration when administered on a QWx3 schedulevia a 1-hour infusion. Observed dose-related toxicities for both specieswere limited to the bone marrow and gastrointestinal tract. These arethe same organs as those observed when free payload (exatecan) wasadministered, suggesting that the delivery of exatecan conjugated to theNDC did not broaden the tissue toxicity profile. Observed toxicitieswere recovered or substantially reduced by the end of a two-weekrecovery period. No drug-related hepatic, renal, pulmonary or oculartoxicities were observed, and there were no drug-related deaths in therepeat dose toxicity study.

TK parameters, estimated in the 15-day GLP study, revealed similarplasma exposure values in males and females for the NDC, total exatecan(conjugated and released) and released exatecan. The NDC exhibited anaverage circulatory half-life ranging from approximately 15 to 20 hoursin rats, and 24 to 29 hours in dogs, with no accumulation of the NDC,total exatecan, or free exatecan observed from day 1 to day 15. Basedupon AUC_(0-last) (hr*ng/mL) released payload levels in the circulationwere less than approximately 0.3% and 0.1% of the total payload levelsin the rat and the dog respectively. No NDC anti-drug antibodies wereinduced in either species. In summary, the NDC has a favorablenonclinical safety/TK profile.

While the invention has been particularly shown and described withreference to specific preferred embodiments, it should be understood bythose skilled in the art that various changes in form and detail may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1-29. (canceled)
 30. A nanoparticle-drug conjugate (NDC) comprising: (a)a silica nanoparticle; (b) polyethylene glycol (PEG) that is covalentlybonded to the surface of the nanoparticle; (c) a targeting ligandcomprising folic acid, wherein the targeting ligand is attached to thenanoparticle through a spacer group; and (d) a linker-payload conjugate,wherein: (i) the payload is exatecan; (ii) the linker-payload conjugateis attached to the nanoparticle through a spacer group; (iii) the linkeris a protease-cleavable linker; and (iv) the exatecan is released uponcleavage of the linker.
 31. The NDC of claim 30, wherein the NDC has anaverage diameter between about 1 nm and about 10 nm.
 32. The NDC ofclaim 30, wherein the NDC has an average diameter between about 5 nm andabout 8 nm.
 33. The NDC of claim 30, wherein the NDC comprises fromabout 1 to about 30 linker-payload conjugates.
 34. The NDC of claim 30,wherein the NDC comprises from about 1 to about 30 targeting ligands.35. The NDC of claim 30, further comprising a fluorescent compound thatis covalently encapsulated within the nanoparticle.
 36. The NDC of claim35, wherein the fluorescent compound comprises a Cy5 dye.
 37. The NDC ofclaim 30, comprising a structure of Formula (NP):

wherein x is an integer of 0 to 20; the silicon atom is a part of thenanoparticle; and the

adjacent to the triazole moiety denotes a point of attachment to thetargeting ligand or the linker-payload conjugate, either directly orindirectly via a linker or spacer group.
 38. The NDC of claim 30,comprisng a structure of Formula (S-1):

wherein Payload comprises exatecan; Linker comprises aprotease-cleavable linker, and the silicon atom is a part of thenanoparticle.
 39. The NDC of claim 30, comprising a structure of Formula(S-2):

wherein Targeting ligand comprises folic acid; and the silicon atom is apart of the nanoparticle.
 40. The NDC of claim 30, wherein thelinker-payload conjugate comprises a structure of Formula (I):

or a salt thereof, wherein,

denotes a point of attachment to the nanoparticle through a spacergroup; A is Val-Lys; Payload comprises a residue of exatecan, wherein Zis a —NH— group of the exatecan; R¹, R², R³, R⁴, and R⁵ in eachoccurrence is independently hydrogen; X is absent; Y is

wherein the carbonyl in

is bonded to Z; X₁, X₂, X₃, and X₄ are each independently —CH—; and nis
 1. 41. The NDC of claim 30, wherein the protease comprises a serineprotease or a cysteine protease.
 42. A nanoparticle-drug conjugate (NDC)comprising: (a) a silica nanoparticle; (b) polyethylene glycol (PEG)that is covalently bonded to the surface of the nanoparticle; (c) a Cy5dye that is covalently encapsulated within the nanoparticle; (d) atargeting ligand that is attached to the nanoparticle through a spacergroup, wherein the targeting ligand is folic acid; (e) a linker-payloadconjugate that is attached to the nanoparticle through a spacer group;wherein the linker-payload conjugate comprises

and wherein the NDC has an average diameter between 1 nm and 10 nm. 43.A nanoparticle drug conjugate (NDC) comprising (a) a silica nanoparticle(b) polyethylene glycol (PEG) that is covalently bonded to the surfaceof the nanoparticle; (c) a structure of Formula (NP-3):

wherein the silicon atom is a part of the nanoparticle; and (d) astructure of Formula (NP-2):

wherein the silicon atom is a part of the nanoparticle.
 44. The NDC ofclaim 43, further comprising a fluorescent dye that is covalentlyencapsulated within the nanoparticle.
 45. A method of treating a cancer,comprising administering to a subject in need thereof an effectiveamount of an NDC of claim
 30. 46. The method of claim 45, wherein thecancer is selected from the group consisting of ovarian cancer,endometrial cancer, fallopian tube cancer, cervical cancer, breastcancer, lung cancer, mesothelioma, uterine cancer, gastrointestinalcancer, pancreatic cancer, bladder cancer, kidney cancer, liver cancer,head and neck cancer, brain cancer, thyroid cancer, skin cancer,prostate cancer, testicular cancer, acute myeloid leukemia (AML), andchronic myelogenous leukemia (CML).
 47. The method of claim 46, whereinthe AML is pediatric AML.
 48. The method of claim 46, wherein the NDC isadministered to the subject intravenously.
 49. A method of treating acancer, comprising administering to a subject in need thereof aneffective amount of an NDC of claim
 42. 50. The method of claim 49,wherein the cancer is selected from the group consisting of ovariancancer, endometrial cancer, fallopian tube cancer, cervical cancer,breast cancer, lung cancer, mesothelioma, uterine cancer,gastrointestinal cancer, pancreatic cancer, bladder cancer, kidneycancer, liver cancer, head and neck cancer, brain cancer, thyroidcancer, skin cancer, prostate cancer, testicular cancer, acute myeloidleukemia (AML), and chronic myelogenous leukemia (CML).
 51. The methodof claim 50, wherein the AML is pediatric AML.
 52. A method of treatinga cancer, comprising administering to a subject in need thereof aneffective amount of an NDC of claim
 43. 53. The method of claim 52,wherein the cancer is selected from the group consisting of ovariancancer, endometrial cancer, fallopian tube cancer, cervical cancer,breast cancer, lung cancer, mesothelioma, uterine cancer,gastrointestinal cancer, pancreatic cancer, bladder cancer, kidneycancer, liver cancer, head and neck cancer, brain cancer, thyroidcancer, skin cancer, prostate cancer, testicular cancer, acute myeloidleukemia (AML), and chronic myelogenous leukemia (CML).
 54. The methodof claim 53, wherein the AML is pediatric AML.
 55. A pharmaceuticalcomposition comprising an NDC of claim 30, and a pharmaceuticallyacceptable excipient.
 56. The pharmaceutical composition of claim 55,wherein the pharmaceutical composition is for intravenousadministration.
 57. A pharmaceutical composition comprising an NDC ofclaim 42, and a pharmaceutically acceptable excipient.
 58. Apharmaceutical composition comprising an NDC of claim 43, and apharmaceutically acceptable excipient.